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  • 1
    Keywords: measurement ; CELLS ; IN-VITRO ; CELL ; MODEL ; DENSITY ; imaging ; HYBRIDIZATION ; RNA ; SACCHAROMYCES-CEREVISIAE ; transcription ; DNA ; DOMAIN ; DYNAMICS ; SIMULATION ; BINDING ; chromosome ; IN-SITU ; CHROMATIN ; YEAST ; REQUIRES ; DERIVATIVES ; FIBER ; LENGTH ; FLUORESCENCE ; FLEXIBILITY ; INTERPHASE ; CHROMATIN STRUCTURE ; DOMAINS ; CHROMOSOMES ; MASSES ; in situ hybridization ; GFP ; 30-nm fiber ; EXTENSION ; HIGH-RESOLUTION ; higher-order structure ; IMAGING TECHNIQUES ; LINKER HISTONE ; nucleosomes
    Abstract: Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of approximate to110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase 11 requires a very transient unfolding of higher-order chromatin structure
    Type of Publication: Journal article published
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  • 2
    Keywords: PEPTIDE ; MODEL ; SITE ; MOLECULES ; MECHANISM ; ANTIGEN ; BINDING ; SEQUENCE ; SEQUENCES ; MOLECULE ; ACTIVE-SITE ; antigen presentation ; PEPTIDES ; LENGTH ; DEGRADATION ; EPITOPES ; antigen processing ; INTERFERON-GAMMA ; ENDOPLASMIC-RETICULUM ; RESIDUES ; TRANSPORTER ; LONG ; endoplasmic reticulum ; ARGININE AMINOPEPTIDASE ; CELL-PROTEINS ; LEUCINE AMINOPEPTIDASE ; LEUKOTRIENE-A(4) HYDROLASE ; PRESENTED PEPTIDES ; proteases ; THIMET OLIGOPEPTIDASE
    Abstract: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an IFN-gamma-induced aminopeptidase in the endoplasmic reticulum that trims longer precursors to the antigenic peptides presented on MHC class I molecules. We recently reported that purified ERAP1 trimmed N-extended precursors but spared peptides of 8 - 9 residues, the length required for binding to MHC class I molecules. Here, we show another remarkable property of ERAP1: that it strongly prefers substrates 9 - 16 residues long, the lengths of pepticles transported efficiently into the ER by the transporter associated with antigen processing (TAP) transporter. This aminopeptidase rapidly degraded a model 13-mer to a 9-mer and then stopped, even though the substrate and the product had identical N- and C-terminal sequences. No other aminopeptidase, including the closely related EIR-aminopeptidase ERAP2, showed a similar length preference. Unlike other aminopeptidases, the activity of ERAP1 depended on the C-terminal residue of the substrate. ERAP1, like most MHC class I molecules, prefers pepticles with hydrophobic C termini and shows low affinity for peptides with charged C termini. Thus, ERAP1 is specialized to process precursors transported by TAP to pepticles that can serve as MHC class I epitopes. Its "molecular ruler" mechanism involves binding the hydrophobic C terminus of the substrate 9 -16 residues away from the active site
    Type of Publication: Journal article published
    PubMed ID: 16286653
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  • 3
    Keywords: brain ; RECEPTOR ; CELLS ; PATHWAY ; PATHWAYS ; GENE ; GENES ; RELEASE ; RESPONSES ; MECHANISM ; FREQUENCY ; hormone ; STRESS ; inactivation ; SIGNALING PATHWAY ; SIGNALING PATHWAYS ; glucocorticoid receptor ; LIVING CELLS ; RECEPTORS ; GLUCOCORTICOID-RECEPTOR ; ANTAGONIST ; rodent ; SUBCELLULAR-LOCALIZATION ; signaling ; NEURONS ; LIFE ; ENHANCEMENT ; ESTROGEN ; corticosteroid ; mineralocorticoid receptor ; LEVEL ; function ; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor CA1 hippocampus ; glucocorticoid receptor knockout ; MICRODIALYSIS ; mineralocorticoid receptor knockout ; miniature excitatory postsynaptic current ; RAT HIPPOCAMPUS ; SYNAPSES
    Abstract: The adrenal hormone corticosterone transcriptionally regulates responsive genes in the rodent hippocampus through nuclear mineralocorticoid and glucocorticoid receptors. Via this genomic pathway the hormone alters properties of hippocampal cells slowly and for a prolonged period. Here we report that corticosterone also rapidly and reversibly changes hippocampal signaling. Stress levels of the hormone enhance the frequency of miniature excitatory postsynaptic potentials in CA1 pyramidal neurons and reduce paired-pulse facilitation, pointing to a hormone-dependent enhancement of glutamate-release probability. The rapid effect by corticosterone is accomplished through a nongenomic pathway involving membrane-located receptors. Unexpectedly, the rapid effect critically depends on the classical mineralocorticoid receptor, as evidenced by the effectiveness of agonists, antagonists, and brain-specific inactivation of the mineralocorticoid but not the glucocorticoid receptor gene. Rapid actions by corticosterone would allow the brain to change its function within minutes after stress-induced elevations of corticosteroid levels, in addition to responding later through gene-mediated signaling pathways
    Type of Publication: Journal article published
    PubMed ID: 16361444
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  • 4
    Keywords: CELLS ; EXPRESSION ; Germany ; CLONING ; GENE ; PROTEIN ; PROTEINS ; transcription ; RELEASE ; DNA ; FAMILY ; DOMAIN ; CARCINOGENESIS ; CONTRAST ; PARTICLES ; virus ; DEGRADATION ; REPLICATION ; ANTIVIRAL ACTIVITY ; CONSTRUCTION ; IMMUNODEFICIENCY-VIRUS ; RE ; spumaretrovirus ; zoonosis ; cytidine deamination ; HIV-1 VIF ; HYPERMUTATION ; restriction factor ; virion infectivity factor
    Abstract: Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet-deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counteracting proteins
    Type of Publication: Journal article published
    PubMed ID: 15911774
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  • 5
    Keywords: CANCER ; KINASE ; SITE ; SITES ; PROTEIN ; ACTIVATION ; DNA ; MECHANISM ; INDUCTION ; mechanisms ; PHOSPHORYLATION ; PROTEIN-KINASE ; LESIONS ; STRESS ; p53 ; DAMAGE ; SIGNALING PATHWAYS ; DNA-DAMAGE ; DEGRADATION ; MDM2 ; DOUBLE-STRAND BREAKS ; ATAXIA-TELANGIECTASIA ; signaling ; RE ; DNA damage ; KINASES ; ATM ; DNA damage response ; protein degradation
    Abstract: Maintenance of genomic stability depends on the DNA damage response, an extensive signaling network that is activated by DNA lesions such as double-strand breaks (DSBs). The primary activator of the mammalian DSB response is the nuclear protein kinase ataxia-telangiectasia, mutated (ATM), which phosphorylates key players in various arms of this network. The activation and stabilization of the p53 protein play a major role in the DNA damage response and are mediated by ATM-dependent posttranslational modifications of p53 and Mdm2, a ubiquitin ligase of p53. p53's response to DNA damage also depends on Mdm2-dependent proteolysis of Mdmx, a homologue of Mdm2 that represses p53's transactivation function. Here we show that efficient damage-induced degradation of human Hdmx depends on functional ATM and at least three sites on the Hdmx that are phosphorylated in response to DSBs. One of these sites, S403, is a direct ATM target. Accordingly, each of these sites is important for Hdm2-mediated ubiquitination of Hdmx after DSB induction. These results demonstrate a sophisticated mechanism whereby ATM fine-tunes the optimal activation of p53 by simultaneously modifying each player in the process
    Type of Publication: Journal article published
    PubMed ID: 15788536
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  • 6
    Keywords: CELLS ; CELL ; Germany ; MICROSCOPY ; PATHWAY ; imaging ; PROTEIN ; SAMPLES ; RESOLUTION ; REDUCTION ; CYCLE ; SIGNAL ; RELAXATION ; PATTERNS ; NUMBER ; NUCLEUS ; MAMMALIAN-CELLS ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; FLUORESCENCE ; ORGANIZATION ; DYE ; ELIMINATION ; intermediate filament ; INCREASE ; FLUORESCENCE MICROSCOPY ; LIFE ; NUCLEAR ; STIMULATED-EMISSION ; DEPLETION ; EXCITATION ; neuron ; BARRIER ; BREAKING ; endosome ; FILAMENTS ; IMPROVEMENT ; photobleaching ; PHOTON ; STED MICROSCOPY ; stimulated emission depletion illumination ; subdiffraction ; triplet state
    Abstract: We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximate to 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences
    Type of Publication: Journal article published
    PubMed ID: 16864773
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  • 7
    Keywords: CELLS ; IN-VITRO ; CELL ; human ; MICROSCOPY ; MODEL ; MODELS ; PATHWAY ; VITRO ; DISEASE ; DISTINCT ; PROTEIN ; PROTEINS ; mechanisms ; DYNAMICS ; polymorphism ; OLIGOMERS ; cytoskeleton ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; vimentin ; REVEALS ; DIMER ; electron microscopy ; intermediate filament ; ELECTRON-MICROSCOPY ; assembly ; PH ; HUMAN VIMENTIN ; analytical ultracentrifugation ; KERATIN FILAMENTS ; BIOLOGICAL MACROMOLECULES ; DIMERS ; AXES ; ADJACENT ; FILAMENTS ; 3D structure
    Abstract: intermediate filaments (IF's), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris(.)HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A(11) mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section
    Type of Publication: Journal article published
    PubMed ID: 17050693
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  • 8
    Keywords: brain ; RECEPTOR ; ANGIOGENESIS ; APOPTOSIS ; CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; INVASION ; proliferation ; SURVIVAL ; tumor ; TUMOR-CELLS ; Germany ; human ; IN-VIVO ; INHIBITION ; KINASE ; THERAPY ; VITRO ; DISTINCT ; TUMORS ; validation ; LINES ; MICE ; TIME ; IDENTIFICATION ; METASTASIS ; CELL-LINE ; leukemia ; LINE ; MIGRATION ; OVEREXPRESSION ; CANCER-THERAPY ; GLIOMAS ; INVITRO ; CELL-GROWTH ; signaling ; BRAIN-TUMORS ; GLIOMA ; PROTOONCOGENE ; LEVEL ; cell migration ; in vivo ; GLIOBLASTOMA ; receptor tyrosine kinase ; GAS6
    Abstract: Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, AxI is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition 〉 85%, P 〈 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, 〉 72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P 〈 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P 〈 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors
    Type of Publication: Journal article published
    PubMed ID: 16585512
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  • 9
    Keywords: A3galt2, ALPHA-CHAIN, antigen presentation, CD1d, dendritic cell, EXPRESSION, GAL-ALPHA(1,3)GAL, gly
    Abstract: CD1d-restricted natural killer T(NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by V alpha 14-J alpha 18 gene segments in mice and V alpha 24-J alpha 18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, hostdefense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4+/ CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrilhexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha 1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR V beta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.
    Type of Publication: Journal article published
    PubMed ID: 17372206
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  • 10
    Keywords: PEPTIDE ; CELLS ; EXPRESSION ; IN-VITRO ; CELL ; Germany ; INHIBITION ; KINASE ; TOXICITY ; VITRO ; DEATH ; PROTEIN ; MICE ; RELEASE ; DOMAIN ; BINDING ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; MOLECULE ; ALPHA ; TARGET ; virus ; CELL-DEATH ; MUTATION ; FUSION ; MUTATIONS ; PEPTIDES ; POLYPEPTIDE ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; TARGET-CELLS ; MINUTE VIRUS ; REPLICATIVE FUNCTIONS ; PROTEIN-PROTEIN INTERACTIONS ; specificity ; LIVING CELLS ; mutagenesis ; SUBSTRATE-SPECIFICITY ; EFFECTOR ; NONSTRUCTURAL PROTEINS ; CYTOTOXICITY ; intermediate filament ; PROGRAM ; PATTERN ; CAPACITY ; parvovirus ; REARRANGEMENT ; DESTRUCTION ; VIRIONS ; SUBSTRATE ; cell death ; USA ; function ; parvovirus minute virus of mice ; FILAMENTS ; PROTECTS ; host ; tropomyosin ; PARVOVIRUS MINUTE VIRUS ; NS1 ; protein kinase specificity
    Abstract: Autonomous parvoviruses induce severe morphological and physiological alterations in permissive host cells, eventually leading to cell lysis and release of progeny virions. Viral cytopathic effects (CPE) result from specific rearrangements and destruction of cytoskeletal micro- and intermediate filaments. We recently reported that inhibition of endogenous casein kinase 11 (CKII) protects target cells from parvovirus minute virus of mice (MVM)-induced CPE, pointing to this kinase as an effector of MVM toxicity. The present work shows that the parvoviral NS1 protein mediates CKII-dependent cytoskeletal alterations and cell death. NS1 can act as an adaptor molecule, linking the cellular protein kinase CK11 alpha to tropomyosin and thus modulating the substrate specificity of the kinase. This action results in an altered tropomyosin phosphorylation pattern both in vitro and in living cells. The capacity of NS1 to induce CPE was impaired by mutations abolishing binding with either CKII alpha or tropomyosin. The cytotoxic adaptor function of NS1 was confirmed with fusion peptides, where the tropomyosin-binding domain of NS1 and CK11a are physically linked. These adaptor peptides were able to mimic NS1 in its ability to induce death of transformed MVM-permissive cells
    Type of Publication: Journal article published
    PubMed ID: 17636126
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