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Sonstige beteiligte Institutionen
Cross-striated muscles enable higher animals to perform directed movements and to create mechanical force. The cells of heart and skeletal muscles consist of myofibrils, serial arrays of the smallest contractile subunits, the sarcomeres. Main components of the sarcomeres are the thin and thick filaments, large protein assemblies consisting of mainly actin (thin filaments) and myosin (thick filaments), whose energy-dependent interaction is responsible for the contraction of sarcomeres and so of the whole muscle. The thin filaments are anchored in the sarcomere bordering Z-discs, while the thick filaments are anchored in the M-bands, traverse structures in the sarcomere center. Electron-microscopic studies revealed that the M-bands consist of regular, lattice-like structures that appear to cross-link the thick filaments. A number of proteins could be identified by immune-fluorescence and biochemical binding studies to be present and interact with each other in the M-bands. These data have been integrated into preliminary models of the M-bands. Detailed knowledge of how these proteins interact with each other in the center of the sarcomeres is, however, largely missing. The current study focuses on the structural characterization of the interactions between the titin, myomesin-1, obscurin and obscurin-like 1 (OBSL1), modular filamentous proteins interacting with each other in the M-bands. The high-resolution crystal structure of the titin M10 – OBSL1 Ig1 complex was solved. The structure and additional biophysical data show that titin and OBSL1 as well as titin and obscurin form stable binary complexes through the formation of a small intermolecular ß-sheet. In contrast to previously characterized intermolecular assemblies of sarcomeric proteins, this sheet is formed between parallel non- homologous ß-strands of the interaction partners. The investigation of disease-related variants of the M10 domain by biophysical methods did not allow to draw unambiguous conclusions on a direct connection between impaired OBSL1/obscurin binding and disease development. Two out of four known M10 variants have effects on the correct domain folding and so interfere with the ability to bind obscurin/OBSL1. The two other known variants displayed however only minor effects on fold and binding affinities. It should therefore be further elucidated whether a direct connection between impaired complex formation and disease development exists. -I- Abstract A direct interaction between titin and myomesin-1 could not be confirmed in vitro. Possible explanations for the different results are discussed. While the consequences of the inability of both proteins to interact are unclear, the further characterization of the putative interacting parts of titin and myomesin-1 led to the discovery of two new potential sites of self-assembly on M-band titin and myomesin-1. The crystal structure of titin M4 showed that this domain can form dimeric assemblies through the formation of a disulfide bridge and an intermolecular metal binding site between residues that are unique to this domain. On myomesin-1, in addition to the described C-terminal interaction site, a potential second site of self-assembly was found in its central Fn3-domain segment. The interacting site was mapped to the predicted Fn3 domain My5. The crystal structure of the domain in its dimeric form showed that the interaction is mediated by a mechanism that has previously not been observed in sarcomeric proteins. Two My5 interact with each other by the mutual exchange of an N-terminal ß-strand which complements the Fn3 fold on the binding partner. This type of interaction can be interpreted as misfolding. However, the position of the interacting domain and its mode of interaction allowed the postulation of a model of how myomesin-1 could be integrated in the M-bands. This model is in good agreement with the electron-microscopic appearance of the M-bands.
Type 1 diabetes affects around 0.5% of the population in developed countries and the incidence rates have been rising over the years. The destruction of beta cells is irreversible and the current therapy available to patients only manages the symptoms and does not prevent the associated pathological manifestations. The patients need lifelong therapy and intensive research is being carried out to identify ways to eliminate autoimmune responses directed against pancreatic beta cells and to replace or regenerate beta cells. The work presented herein aimed at analyzing the role of the Th17 T cell subset, characterized by secretion of the pro- inflammatory cytokine IL-17A, in autoimmune diabetes and also at generating a beta cell reporter mouse line in the NOD background, the most widely- used mouse model for type 1 diabetes. We generated IL- 17A knockdown (KD) NOD mice, using RNAi in combination with lentiviral transgenesis. We analyzed diabetes frequency in IL-17A deficient mice and found that the loss of IL-17A did not protect the transgenic mice from diabetes. Based on these observations, we believe that Th17 cells do not play a critical role in type 1 diabetes through the IL-17A pathway, though they might still be involved in the disease process through alternate pathways. We also generated NOD and NOD-SCID mice with a transgene that drives the beta cell specific expression of a luciferase reporter gene. We used a lentiviral construct, which combined a luciferase sequence and a short- hairpin RNA (shRNA) expression cassette, allowing gene- knockdown under the beta cell specific rat insulin promoter (RIP). These mice will be of use in studying beta cell phenotypes resulting from the knockdown of target genes, using non- invasive bioimaging. We believe that the generation of these reporter mouse lines for diabetes studies will prove valuable in future investigations. Furthermore, the demonstration that the loss of IL-17A does not alter susceptibility to type 1 diabetes should help clarify the controversial involvement of Th17 cells in this disease.
Background:
Inhibition of early platelet adhesion by blockade of glycoprotein-IB (GPIb) protects mice from ischemic stroke. To elucidate underlying mechanisms in-vivo, infarct development was followed by ultra-high field MRI at 17.6 Tesla.
Methods:
Cerebral infarction was induced by transient-middle-cerebral-artery-occlusion (tMCAO) for 1 hour in C57/BL6 control mice (N = 10) and mice treated with 100 mg Fab-fragments of the GPIb blocking antibody p0p/B 1 h after tMCAO (N = 10). To control for the effect of reperfusion, additional mice underwent permanent occlusion and received anti-GPIb treatment (N = 6; pMCAO) or remained without treatment (N = 3; pMCAO). MRI 2 h and 24 h after MCAO measured cerebral-blood-flow (CBF) by continuous arterial-spin labelling, the apparent-diffusion-coefficient (ADC), quantitative-T2 and T2-weighted imaging. All images were registered to a standard mouse brain MRI atlas and statistically analysed voxel-wise, and by cortico-subcortical ROI analysis.
Results:
Anti-GPIb treatment led to a relative increase of postischemic CBF vs. controls in the cortical territory of the MCA (2 h: 44.2 +/- 6.9 ml/100g/min versus 24 h: 60.5 +/- 8.4; p = 0.0012, F((1,18)) = 14.63) after tMCAO. Subcortical CBF 2 h after tMCAO was higher in anti-GPIb treated animals (45.3 +/- 5.9 vs. controls: 33.6 +/- 4.3; p = 0.04). In both regions, CBF findings were clearly related to a lower probability of infarction (Cortex/Subcortex of treated group: 35%/65% vs. controls: 95%/100%) and improved quantitative-T2 and ADC. After pMCAO, anti-GPIb treated mice developed similar infarcts preceded by severe irreversible hypoperfusion as controls after tMCAO indicating dependency of stroke protection on reperfusion.
Conclusion:
Blockade of platelet adhesion by anti-GPIb-Fab-fragments results in substantially improved CBF early during reperfusion. This finding was in exact spatial correspondence with the prevention of cerebral infarction and indicates in-vivo an increased patency of the microcirculation. Thus, progression of infarction during early ischemia and reperfusion can be mitigated by anti-platelet treatment.
Background:
The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA.
Results:
We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo.
Conclusions:
We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.
Switches in trypanosome differentiation: ALBA proteins acting on post-transcriptional mRNA control
(2011)
Trypanosoma brucei is a digenetic eukaryotic parasite that develops in different tissues of a mammalian host and a tsetse fly. It is responsible for sleeping sickness in sub-saharan Africa. The parasite cycle involves more than nine developmental stages that can be clearly distinguished by their general morphology, their metabolism and the relative positioning of their DNA-containing organelles. During their development, trypanosomes remain exclusively extracellular and encounter changing environments with different physico-chemical properties (nutritional availability, viscosity, temperature, etc.). It has been proposed that trypanosomes use their flagellum as a sensing organelle, in agreement with the established role of structurally-related cilia in metazoa and ciliates. Recognition of environmental triggers is presumed to be at the initiation of differentiation events, leading to the parasite stage that is the best suited to the new environment. These changes are achieved by the modification of gene expression programmes, mostly underlying post-transcriptional control of mRNA transcripts. We first demonstrate that the RNA-binding proteins ALBA3/4 are involved in specific differentiation processes during the parasite development in the fly. They are cytosolic and expressed throughout the parasite cycle with the exception of the stages found in the tsetse fly proventriculus, as shown by both immunofluorescence and live cell analysis upon endogenous tagging with YFP. Knock-down of both proteins in the developmental stage preceding these forms leads to striking modifications: cell elongation, cell cycle arrest and relocalization of the nucleus in a posterior position, all typical of processes acting in parasites found in the proventriculus region. When ALBA3 is over-expressed from an exogenous copy during infection, it interferes with the relocalization of the nucleus in proventricular parasites. This is not observed for ALBA4 over-expression that does not visibly impede differentiation. Both ALBA3/4 proteins react to starvation conditions by accumulating in cytoplasmic stress granules together with DHH1, a recognized RNA-binding protein. ALBA3/4 proteins also partially colocalize with granules formed by polyA+ RNA in these conditions. We propose that ALBA are involved in trypanosome differentiation processes where they control a subset of developmentally regulated transcripts. These processes involving ALBA3/4 are likely to result from the specific activation of sensing pathways. In the second part of the thesis, we identify novel flagellar proteins that could act in sensing mechanisms. Several protein candidates were selected from a proteomic analysis of intact flagella performed in the host laboratory. This work validates their flagellar localization with high success (85% of the proteins examined) and defines multiple different patterns of protein distribution in the flagellum. Two proteins are analyzed during development, one of them showing down-regulation in proventricular stages. The functional analysis of one novel flagellar membrane protein reveals its rapid dynamics within the flagellum but does not yield a visible phenotype in culture. This is coherent with sensory function that might not be needed in stable culture conditions, but could be required in natural conditions during development. In conclusion, this work adds new pieces to the puzzle of identifying molecular switches involved in developmental mRNA control and environmental sensing in trypanosome stages in the tsetse fly.
Desert ants of the genus Cataglyphis have become model systems for the study of insect navigation. An age-related polyethism subdivides their colonies into interior workers and short-lived light-exposed foragers. While foraging in featureless and cluttered terrain over distances up to several hundred meters, the ants are able to precisely return back to their often inconspicuous nest entrance. They accomplish this enormous navigational performance by using a path integration system - including a polarization compass and an odometer - as their main navigational means in addition to landmark-dependent orientation and olfactory cues. C. fortis, being the focus of the present thesis, is endemic to the salt flats of western North Africa, which are completely avoided by other Cataglyphis species. The fact that Cataglyphis ants undergo a behavioral transition associated with drastically changing sensory demands makes these ants particularly interesting for studying synaptic plasticity in visual and olfactory brain centers. This thesis focuses on plastic changes in the mushroom bodies (MBs) - sensory integration centers supposed to be involved in learning and memory presumably including landmark learning - and in synaptic complexes belonging to the lateral accessory lobe (LAL) known to be a relay station in the polarization processing pathway. To investigate structural synaptic plasticity in the MBs of C. fortis, synaptic complexes (microglomeruli, MG) in the visual (collar) and olfactory (lip) input regions of the MB calyx were immunolabeled and their pre- and postsynaptic profiles were quantified. The results show that a volume increase of the MB calyx during behavioral transition is associated with a decrease of MG number - an effect called pruning - in the collar and, less pronounced, in the lip that goes along with dendritic expansion in MB intrinsic Kenyon cells. Light-exposure of dark-reared ants of different age classes revealed similar effects and dark-reared ants age-matched to foragers had MG numbers comparable to those of interior workers. The results indicate that the enormous structural synaptic plasticity of the MB calyx collar is primarily driven by visual experience rather than by an internal program. Ants aged artificially for up to one year expressed a similar plasticity indicating that the system remains flexible over the entire life-span. To investigate whether light-induced synaptic reorganization is reversible, experienced foragers were transferred back to darkness with the result that their MBs exhibit only some reverse-type characteristics, in particular differences in presynaptic synapsin expression. To investigate the structure of large synaptic complexes in the LAL of C. fortis and to detect potential structural changes, pre- and postsynaptic profiles in interior workers and foragers were immunolabeled and quantified by using confocal imaging and 3D-reconstruction. The results show that these complexes consist of postsynaptic processes located in a central region that is surrounded by a cup-like presynaptic profile. Tracer injections identified input and output tracts of the LAL: projection neurons from the anterior optic tubercle build connections with neurons projecting to the central complex. The behavioral transition is associated with an increase by ~13% of synaptic complexes suggesting that the polarization pathway may undergo some sort of calibration process. The structural features of these synaptic contacts indicate that they may serve a fast and reliable signal transmission in the polarization vision pathway. Behavioral analyses of C. fortis in the field revealed that the ants perform exploration runs including pirouette-like turns very close to the nest entrance for a period of up to two days, before they actually start their foraging activity. During these orientation runs the ants gather visual experience and might associate the nest entrance with specific landmarks or get entrained to other visual information like the polarization pattern, and, concomitantly adapt their neuronal circuitries to the upcoming challenges. Moreover, the pirouettes may serve to stimulate and calibrate the neuronal networks involved in the polarization compass pathway. Video recordings and analyses demonstrate that light experience enhanced the ants’ locomotor activity after three days of exposure. The fact that both the light-induced behavioral and neuronal changes in visual brain centers occur in the same time frame suggests that there may be a link between structural synaptic plasticity and the behavioral transition from interior tasks to outdoor foraging. Desert ants of the genus Cataglyphis possess remarkable visual navigation capabilities, but also employ olfactory cues for detecting nest and food sites. Using confocal imaging and 3D-reconstruction, potential adaptations in primary olfactory brain centers were analyzed by comparing the number, size and spatial arrangement of olfactory glomeruli in the antennal lobe of C. fortis, C. albicans, C. bicolor, C. rubra, and C. noda. Workers of all Cataglyphis species have smaller numbers of glomeruli compared to those of more olfactory-guided Formica species - a genus closely related to Cataglyphis - and to those previously found in other olfactory-guided ant species. C. fortis has the lowest number of glomeruli compared to all other species, but possesses a conspicuously enlarged glomerulus that is located close to the antennal nerve entrance. Males of C. fortis have a significantly smaller number of glomeruli compared to female workers and queens and a prominent male-specific macroglomerulus likely to be involved in sex pheromone communication. The behavioral significance of the enlarged glomerulus in female workers remains elusive. The fact that C. fortis inhabits microhabitats that are avoided by all other Cataglyphis species suggests that extreme ecological conditions may not only have resulted in adaptations of visual capabilities, but also in specializations of the olfactory system. The present thesis demonstrates that Cataglyphis is an excellent candidate for studying the neuronal mechanisms underlying navigational features and for studying neuronal plasticity associated with the ant’s lifelong flexibility of individual behavioral repertoires.
Background: To perform a systematic review and meta-analysis on cytokine levels in patients with fibromyalgia syndrome (FMS). Methods: Through December 2010 we systematically reviewed the databases PubMed, MEDLINE, and PsycINFO and screened the reference lists of 22 review articles for suitable original articles. Original articles investigating cytokines in patients with FMS were included. Data were extracted by two independent authors. Differences of the cytokine levels of FMS patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed applying methodological scores: modified Center of Evidence Based Medicine, Newcastle-Ottawa-Scale, and Würzburg Methodological Quality Score. Results: Twenty-five articles were included investigating 1255 FMS patients and 800 healthy controls. Data of 13/25 studies entered meta-analysis. The overall methodological quality of studies was low. The results of the majority of studies were not comparable because methods, investigated material, and investigated target cytokines differed. Systematic review of the selected 25 articles revealed that FMS patients had higher serum levels of interleukin (IL)-1 receptor antagonist, IL-6, and IL-8, and higher plasma levels of IL-8. Meta-analysis of eligible studies showed that FMS patients had higher plasma IL-6 levels compared to controls (SMD = -0.34 [-0.64, -0.03] 95% CI; p = 0.03). The majority of investigated cytokines were not different between patients and controls. Conclusions: The pathophysiological role of cytokines in FMS is still unclear. Studies of higher quality and with higher numbers of subjects are needed.
Background:
The use of DNA based methods for assessing biodiversity has become increasingly common during the last years. Especially in speciose biomes as tropical rain forests and/or in hyperdiverse or understudied taxa they may efficiently complement morphological approaches. The most successful molecular approach in this field is DNA barcoding based on cytochrome c oxidase I (COI) marker, but other markers are used as well. Whereas most studies aim at identifying or describing species, there are only few attempts to use DNA markers for inventorying all animal species found in environmental samples to describe variations of biodiversity patterns.
Methodology/Principal Findings:
In this study, an analysis of the nuclear D3 region of the 28S rRNA gene to delimit species-like units is compared to results based on distinction of morphospecies. Data derived from both approaches are used to assess diversity and composition of staphylinid beetle communities of a Guineo-Congolian rain forest in Kenya. Beetles were collected with a standardized sampling design across six transects in primary and secondary forests using pitfall traps. Sequences could be obtained of 99% of all individuals. In total, 76 molecular operational taxonomic units (MOTUs) were found in contrast to 70 discernible morphospecies. Despite this difference both approaches revealed highly similar biodiversity patterns, with species richness being equal in primary and secondary forests, but with divergent species communities in different habitats. The D3-MOTU approach proved to be an efficient tool for biodiversity analyses.
Conclusions/Significance:
Our data illustrate that the use of MOTUs as a proxy for species can provide an alternative to morphospecies identification for the analysis of changes in community structure of hyperdiverse insect taxa. The efficient amplification of the D3-marker and the ability of the D3-MOTUs to reveal similar biodiversity patterns as analyses of morphospecies recommend its use in future molecular studies on biodiversity.
Testing Pollen of Single and Stacked Insect-Resistant Bt-Maize on In vitro Reared Honey Bee Larvae
(2011)
The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.
We herein present a patient with delayed-type allergic hypersensitivity against prilocaine leading to spreading eczematous dermatitis after subcutaneous injections for local anesthesia with prilocaine. Prilocaine allergy was proven by positive skin testing and subcutaneous provocation, whereas the evaluation of other local anesthetics - among them lidocaine, articaine and mepivacaine - did not exhibit any evidence for cross-reactivity. Interestingly, our patient repeatedly tolerated strictly deep subcutaneous injection of prilocaine in provocation testing while patch and superficial subcutaneous application mounted strong allergic responses. We hypothesize, that lower DC density in deeper cutaneous compartments and/or different DC subsets exhibiting distinct functional immunomodulatory properties in the various layers of the skin may confer to the observed absence of clinical reactivity against prilocaine after deep subcutaneous injection. The term compartment allergy indicates that the route of allergen administration together with the targeted immunologic environment orchestrates on the immunologic outcome: overt T-cell mediated allergy or clinical tolerance.