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Research Interests: Stem Cells, Confocal Microscopy, Molecular endocrinology, Fluorescence Resonance Energy Transfer, Western blotting, and 11 moreBiological Sciences, Hippocampus, Cercopithecus aethiops, Mutation, Animals, Polymerase Chain Reaction, Neurons, Immunoprecipitation, Cell nucleus, Rats, and Protein Binding
Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation... more
Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.
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ABSTRACT Recently, we have established zebrafish as a novel exercise model and demonstrated the stimulation of growth by exercise. Exercise may also induce cardiac hypertrophy and cardiomyocyte proliferation in zebrafish making it an... more
ABSTRACT Recently, we have established zebrafish as a novel exercise model and demonstrated the stimulation of growth by exercise. Exercise may also induce cardiac hypertrophy and cardiomyocyte proliferation in zebrafish making it an important model to study vertebrate heart regeneration and improved robustness of fish in aquaculture. Moreover, zebrafish has been recently recognized as a valuable model for immunological studies since they possess an adaptive and innate immune system similar to mammals. Given the beneficial effects of exercise on mammalian immune function, zebrafish can be used to investigate exercise-induced stimulation of immune function. Current studies are aimed to demonstrate the mechanisms behind the beneficial exercise effects of enhanced skeletal and cardiac muscle growth and immune functioning and to assess the role of the cortisol stress response at the base of these beneficial exercise effects. As such, zebrafish can be used as an exercise model with implications in basic, biomedical, and applied sciences, such as aquaculture
In humans and zebrafish, two glucocorticoid receptor splice variants exist: the canonical GR α-isoform (GRα), and the GR β-isoform (GRβ). In the present study we have used the zebrafish model system in order to reveal genes affected by... more
In humans and zebrafish, two glucocorticoid receptor splice variants exist: the canonical GR α-isoform (GRα), and the GR β-isoform (GRβ). In the present study we have used the zebrafish model system in order to reveal genes affected by each of these two receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GR β-isoform. In addition, specific GRβ overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic glucocorticoid dexamethasone and transcriptome analysis was performed. Two distinct gene clusters were found that are regulated by GRα. One that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that is regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRβ may act as a dominant-negative inhibitor of GRα when GRβ is overexpr...
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Auditory sensitivity in fish serves various important functions, but also makes fish susceptible to noise pollution. Human-generated sounds may affect behavioral patterns of fish, both in natural conditions and in captivity. Fish are... more
Auditory sensitivity in fish serves various important functions, but also makes fish susceptible to noise pollution. Human-generated sounds may affect behavioral patterns of fish, both in natural conditions and in captivity. Fish are often kept for consumption in aquaculture, on display in zoos and hobby aquaria, and for medical sciences in research facilities, but little is known about the impact of ambient sounds in fish tanks. In this study, we conducted two indoor exposure experiments with zebrafish (Danio rerio). The first experiment demonstrated that exposure to moderate sound levels (112 dB re 1 μPa) can affect the swimming behavior of fish by changing group cohesion, swimming speed and swimming height. Effects were brief for both continuous and intermittent noise treatments. In the second experiment, fish could influence exposure to higher sound levels by swimming freely between an artificially noisy fish tank (120-140 dB re 1 μPa) and another with ambient noise levels (89 d...
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Alternative mRNA splicing in the region encoding the C-terminus of nuclear receptors results in receptor variants lacking the entire ligand-binding domain (LBD), or a part of it, and instead contain a sequence of splice variant-specific... more
Alternative mRNA splicing in the region encoding the C-terminus of nuclear receptors results in receptor variants lacking the entire ligand-binding domain (LBD), or a part of it, and instead contain a sequence of splice variant-specific C-terminal amino acids. A total of thirteen such splice variants have been shown to occur in vertebrates, and at least nine occur in humans. None of these receptor variants appear to be able to bind endogenous ligands and to induce transcription on promoters containing the response element for the respective canonical receptor variant. Interestingly, ten of these C-terminal splice variants have been shown to display dominant-negative activity on the transactivational properties of their canonical equivalent. Research on most of these splice variants has been limited, and the dominant-negative effect of these receptor variants has only been demonstrated in reporter assays in vitro, using transiently transfected receptors and reporter constructs. Therefore, the in vivo function and relevance of most C-terminal splice variants remains unclear. By reviewing the literature on the human glucocorticoid receptor beta-isoform (hGRbeta), we show that the dominant-negative effect of hGRbeta is well established using more physiologically relevant readouts. The hGR beta-isoform may alter gene transcription independent from the canonical receptor and increased hGRbeta levels correlate with glucocorticoid resistance and the occurrence of several immune-related diseases. Thus, available data suggests that C-terminal splice variants of nuclear receptors act as dominant-negative inhibitors of receptor-mediated signaling in vivo, and that aberrant expression of these isoforms may be involved in the pathogenesis of a variety of diseases.
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Corticosterone and Brain-Derived Neurotrophic Factor (BDNF) have both been shown to be involved in spatial memory formation in rats. In the present study we have investigated the effect of corticosterone on hippocampal BDNF mRNA... more
Corticosterone and Brain-Derived Neurotrophic Factor (BDNF) have both been shown to be involved in spatial memory formation in rats. In the present study we have investigated the effect of corticosterone on hippocampal BDNF mRNA expression after training in the Morris water maze in young adult Wistar rats. Therefore, we first studied BDNF mRNA levels in the hippocampus in relation to corticosterone levels at several time points after 4 training trials in the Morris water maze. Corticosterone levels were significantly increased after this procedure, and hippocampal BDNF mRNA levels only displayed a minor change: an increase in CA1 at 1 hr after training. However, in a previous study we observed dramatically decreased hippocampal BDNF mRNA levels in dentate gyrus and CA1 at 3 hr after injection of corticosterone. In order to analyze this discrepancy, we subsequently investigated if hippocampal BDNF mRNA expression is affected by corticosterone at 3 hr after water maze training. Therefore, we incorporated ADX animals and ADX animals which were injected with corticosterone in our study. ADX animals which were subjected to water maze training displayed similar hippocampal BDNF mRNA levels 3 hr after training compared to control ADX animals. Furthermore, ADX animals which were injected with corticosterone showed decreased BDNF mRNA levels in all hippocampal regions compared to control ADX animals. Water maze training did not alter this effect. Thus, the increased corticosterone levels during water maze training do not affect hippocampal BDNF mRNA expression, although exogenous corticosterone is effective under these conditions. Hence, our results suggest that in this situation BDNF is resistant to regulation by endogenous corticosterone, which may be important for learning and memory processes.
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Research Interests: Rheumatoid Arthritis, STEROIDS, Molecular Mechanics, Cortisol, Humans, and 17 moreMutation, Animals, Drug Resistance, Negative Feedback, HPA axis, Animal Model, Genetic Screening, Behavioral Animal Models, Clinical Sciences, Pituitary Gland, Major Depression, Genetically Modified, Model System, Zebrafish, Physiological Stress Markers, Protein isoforms, and Glucocorticoids
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The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear... more
The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.
Research Interests: Confocal Microscopy, STEROIDS, Molecular endocrinology, Nuclear Receptor, Biological Sciences, and 19 moreMolecular, Software, Humans, Mutagenesis, Mutation, Animals, Plasmids, Cell nucleus, Triamcinolone Acetonide, Photons, Time Factors, Transfection, fluorescence recovery after photobleaching (FRAP), Structure activity Relationship, Protein Binding, Structure Function, Ligands, Cortisone, and Glucocorticoids
Glucocorticoids are compounds that have successfully been used over the years in the treatment of inflammatory disorders. They are known to exhibit their effects through the glucocorticoid receptor (GR) that acts to downregulate the... more
Glucocorticoids are compounds that have successfully been used over the years in the treatment of inflammatory disorders. They are known to exhibit their effects through the glucocorticoid receptor (GR) that acts to downregulate the action of proinflammatory transcription factors such as AP-1 and NF-κB. The GR also exerts anti-inflammatory effects through activation of distinct genes. In addition to their anti-inflammatory actions, glucocorticoids are also potent antiallergic compounds that are widely used in conditions such as asthma and anaphylaxis. Nevertheless the mechanism of action of this hormone in these disorders is not known. In this article, we have reviewed reports on the effects of glucocorticoids in mast cells, one of the important immune cells in allergy. Building on the knowledge of the molecular action of glucocorticoids and the GR in the treatment of inflammation in other cell types, we have made suggestions as to the likely mechanisms of action of glucocorticoids in mast cells. We have further identified some important questions and research directions that need to be addressed in future studies to improve the treatment of allergic disorders.
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Research Interests: Photochemistry, Fluorescent Dyes and Reagents, Confocal Microscopy, Biological Sciences, Cercopithecus aethiops, and 24 moreDexamethasone, Cell line, Molecular and cellular biology, Humans, Kidney, Animals, Corticosterone, Ligand Binding, Cell nucleus, Triamcinolone Acetonide, Protease Inhibitors, HeLa cells, Multienzyme complexes, Cysteine endopeptidases, Transfection, Protein Conformation, Protein isoforms, fluorescence recovery after photobleaching (FRAP), Protein Binding, Cytoplasm, Ligands, Conformational Change, Glucocorticoids, and PROTEASOME INHIBITOR
ABSTRACT Single zebrafish eggs were rapidly profiled using high resolution-direct infusion-nanoelectrospray-mass spectrometry with limited sample preparation and without separation. The analysis time per sample is around 1 min. Using this... more
ABSTRACT Single zebrafish eggs were rapidly profiled using high resolution-direct infusion-nanoelectrospray-mass spectrometry with limited sample preparation and without separation. The analysis time per sample is around 1 min. Using this approach the different developmental stages of zebrafish eggs can be characterized by their active metabolites. Five different development stages with distinct metabolic fingerprints could clearly be observed when untargeted analysis is performed and the data are plotted using principal component analysis. Using this approach early embryogenesis is followed with a time resolution of 1 h and 102 features proved relevant. Of these, significant number of putatively identified compounds has not been reported earlier to have any association with early zebrafish embryogenesis yet. The onset of gene expression and the increase in energy requirement is reflected by the measured metabolome complementing earlier reported transcriptomics studies from a systems biology point of view. By deyolking and dechorionation eggs at two early developmental stages, we were able to observe distinct changes in localized metabolism.
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Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if... more
Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if neurotrophins can be implicated in these effects. For this purpose we injected 30, 300, and 1,000 microg corticosterone s.c. (per kg body weight) in adrenalectomized rats and measured the mRNA levels of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase (trk)B, neurotrophin (NT)-3, and trkC in hippocampal cell fields at 6 hr after steroid administration by in situ hybridization. NT-3 and trkC mRNA did not show significant changes in any hippocampal region after the various doses of corticosterone. BDNF mRNA decreased after corticosterone administration dose dependently, resulting in a maximal suppression of 35, 20, and 50% in dentate gyrus, CA3, and CA1, respectively. Interestingly, trkB responded to corticosterone in an inverted U-shaped fashion in CA3 and dentate gyrus: the low dose of corticosterone increased trkB mRNA expression in both regions by approximately 30%, while the effect of the two higher doses was not different from the vehicle injected controls. In conclusion, we found differential effects of low and high doses of corticosterone on BDNF and trkB expression in hippocampus, which suggests involvement of a coordinated MR- and GR-mediated action.
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Owing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a... more
Owing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a consistent quantitative scheme of their action is still lacking. Using the androgen receptor (AR) as a model system, we combined three different fluorescence microscopy assays: single-molecule microscopy, photobleaching and correlation spectroscopy, to provide a quantitative model of the action of this transcription factor. This approach enabled us to distinguish two types of AR-DNA binding: very brief interactions, in the order of a few hundred milliseconds, and hormone-induced longer-lasting interactions, with a characteristic binding time of several seconds. In addition, freely mobile ARs were slowed down in the presence of hormone, suggesting the formation of large AR-co-regulator complexes in the nucleoplasm upon hormone activation. Our data suggest a model in which mobile hormone-induced complexes of transcription factors and co-regulators probe DNA by briefly binding at random sites, only forming relatively stable transcription initiation complexes when bound to specific recognition sequences.
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Research Interests: Biological Chemistry, Fluorescent Dyes and Reagents, Biological Sciences, DNA, Cell line, and 16 moreHumans, Animals, Biological, Photobleaching, Transcription Factor, CHEMICAL SCIENCES, DNA binding, Acetylation, Transfection, fluorescence recovery after photobleaching (FRAP), Protein Binding, DNA binding proteins, Binding Site, Enzyme Linked Immunosorbent Assay, Protein Transport, and DNA sequence
Research Interests: Breast Cancer, Reproductive Biology, Oogenesis, Signal Transduction, Biological Sciences, and 16 moreEnvironmental Sciences, Progesterone, Humans, Female, Animals, Subcellular Localization, Oocyte Maturation, Cell nucleus, Zebrafish, Ovary, Oocytes, Second Messengers, Intracellular Signaling, Acrosome Reaction, Oocyte, and Breast Cancer Cells
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Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target... more
Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s) and the other half for longer time periods (∼ 2.3 s). A similar pattern of mobility was seen for the MR activated by a...
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Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as... more
Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
Research Interests: Developmental Biology, Tissue Engineering, Immune response, Computational Biology, Pattern Formation, and 17 moreInflammation, Drug development, Biological Sciences, Humans, Automation, High throughput screening, Animals, The, Scaling up, Cost effectiveness, Phenotype, Immune system, Body Patterning, Basic Research, Zebrafish, Amino Acid Sequence, and Embryos
The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects... more
The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects on memory processes are mediated by alterations in the expression of the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus. First, studies are described investigating the effect of corticosterone on BDNF expression in the rat hippocampus. It appears that corticosterone suppresses the BDNF expression at the mRNA and protein level in a subfield-specific way. Second, a model for the mechanism of action is proposed. In this model, activated mineralocorticoid and glucocorticoid receptors repress transcriptional activity of the BDNF promoter site-specifically via interaction with other transcription factors. Third, the implications for learning and memory are discussed. Studies show that during water maze training, corticosterone levels rise significantly, but the BDNF expression is not suppressed in any hippocampal subfield. Furthermore, high BDNF expression levels in specific subfields correlate with a good memory performance. Therefore, we suggest that the resistance of the hippocampal BDNF expression to suppression by corticosterone, as seen after water maze training, may contribute to an optimal memory performance.
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Research Interests: Biomedical Engineering, Kinetics, Fluorescence, Fluorescence Microscopy, Single molecule, and 12 moreBiomedical Optics, Humans, Animals, Optical physics, Protein Complex Detection, Molecules, Optometry and Ophthalmology, Rats, Biological Process, Fluorescent Protein, Membrane Protein, and Plasma Membrane
Research Interests: Genetics, Cell Adhesion, Gene expression, Signal Transduction, Hippocampus, and 14 moreLearning and Memory, Animals, Neuronal Plasticity, Alternative splicing, Rats, Calmodulin, Brain Structure, Feedback loop, Alternative Splicing, Protein Kinase, Differential Display, Mineralocorticoid Receptor, Gene expression profiling, and Differential expression
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Previously, we showed that corticosterone regulates BDNF mRNA levels in the hippocampus. In the present study, we have investigated the time course and dose-dependency of this effect at both the mRNA and the protein level. Corticosterone... more
Previously, we showed that corticosterone regulates BDNF mRNA levels in the hippocampus. In the present study, we have investigated the time course and dose-dependency of this effect at both the mRNA and the protein level. Corticosterone was administered in doses of 30 and 1000 microgram/kg b.w. subcutaneously to adrenalectomized animals. At 3, 6, 12 and 24 h after administration BDNF and trkB mRNA levels in hippocampal subfields were measured by in situ hybridization. Our results show a dose-dependent decrease in BDNF mRNA in dentate gyrus and CA1 at 3 h. After the high dose, this decrease was 70% and 40% respectively. In addition, ELISA was performed to study if this downregulation is also detectable at the protein level. Hippocampal tissue was used from adrenalectomized animals which had received 1000 microgram/kg b.w. corticosterone 4 and 6 h before decapitation. At both time points, a decrease in BDNF protein was observed; 17% at 4 h and 14% at 6 h after corticosterone, as compared to the vehicle injected controls. TrkB mRNA levels were not affected by corticosterone. However, between 6 and 24 h after treatment, increases in trkB mRNA were observed. In conclusion, we have found a transient, dose-dependent decrease in BDNF mRNA and protein in the hippocampus, which may underly changes in neuronal plasticity in the hippocampus after short-term changes in corticosterone concentrations.