Sondersammelgebiets-Volltexte
Refine
Year of publication
- 2008 (10) (remove)
Document Type
- Article (10)
Language
- English (10)
Has Fulltext
- yes (10) (remove)
Is part of the Bibliography
- no (10)
Keywords
Institute
- Medizin (10) (remove)
Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.
Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR γ-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cγ (PLCγ). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5′-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR γ-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.
Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.
Background: The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection. Methods and Findings: Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge. Conclusions: The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.
Poster presentation: Introduction The brain is a highly interconnected network of constantly interacting units. Understanding the collective behavior of these units requires a multi-dimensional approach. The results of such analyses are hard to visualize and interpret. Hence tools capable of dealing with such tasks become imperative. ....
Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection
(2008)
Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6–infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, “fused” liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6–infected mice generated type 1 liver kidney microsomal–like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6–infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.
Transcriptional activation involves the ordered recruitment of coactivators via direct interactions between distinct binding domains and recognition motifs. The p160/SRC/NCoA coactivator family comprises three members (NCoA-1, -2 and -3), which are organized in multiprotein coactivator complexes. We had identified the PAS-B domain of NCoA-1 as an LXXLL motif binding domain. Here we show that NCoA family members are able to interact with other full-length NCoA proteins via their PAS-B domain and they specifically interact with the CBP-interaction domain (CID/AD1) of NCoA-1. Peptide competition, binding experiments and mutagenesis of LXXLL motifs point at distinct binding motif specificities of the NCoA PAS-B domains. NMR studies of different NCoA-1-PAS-B/LXXLL peptide complexes revealed similar although not identical binding sites for the CID/AD1 and STAT6 transactivation domain LXXLL motifs. In mechanistic studies, we found that overexpression of the PAS-B domain is able to disturb the binding of NCoA-1 to CBP in cells and that a CID/AD1 peptide competes with STAT6 for NCoA-1 in vitro. Moreover, the expression of an endogenous androgen receptor target gene is affected by the overexpression of the NCoA-1 or NCoA-3 PAS-B domains. Our study discloses a new, complementary mechanism for the current model of coactivator recruitment to target gene promoters.
Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.
Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor ~18–25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.
Background The EGF receptor has been shown to internalize via clathrin-independent endocytosis (CIE) in a ligand concentration dependent manner. From a modeling point of view, this resembles an ultrasensitive response, which is the ability of signaling networks to suppress a response for low input values and to increase to a pre-defined level for inputs exceeding a certain threshold. Several mechanisms to generate this behaviour have been described theoretically, the underlying assumptions of which, however, have not been experimentally demonstrated for the EGF receptor internalization network. Results Here, we present a mathematical model of receptor sorting into alternative pathways that explains the EGF-concentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrin-dependent endocytosis pathway and implies distinct steady-states into which the system is forced for low vs high EGF stimulations. The model is minimal since no experimentally unjustified reactions or parameter assumptions are imposed. We demonstrate the robustness of the sorting effect for large parameter variations and give an analytic derivation for alternative steady-states that are reached. Further, we describe extensibility of the model to more than two pathways which might play a role in contexts other than receptor internalization. Conclusions Our main result is that a scenario where different endocytosis routes consume the same form of receptor corroborates the observation of a clear-cut, stimulus dependent sorting. This is especially important since a receptor modification discriminating between the pathways has not been found. The model is not restricted to EGF receptor internalization and might account for ultrasensitivity in other cellular contexts.