Subcellular analysis of blood-brain barrier function by micro-impalement of vessels in acute brain slices.

The blood-brain barrier (BBB) is a tightly and actively regulated vascular barrier. Answering fundamental biological and translational questions about the BBB with currently available approaches is hampered by a trade-off between accessibility and biological validity. We report an approach combining micropipette-based local perfusion of capillaries in acute brain slices with multiphoton microscopy.

Optical analysis of glutamate spread in the neuropil.

Fast synaptic communication uses diffusible transmitters whose spread is limited by uptake mechanisms. However, on the submicron-scale, the distance between two synapses, the extent of glutamate spread has so far remained difficult to measure. Here, we show that quantal glutamate release from individual hippocampal synapses activates extracellular iGluSnFr molecules at a distance of >1.

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry.

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics.

Differential regulation of Foxo3a target genes in erythropoiesis.

The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5.

TGF-beta-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen.

Transforming growth factor-beta (TGF-beta), a key modulator of endothelial cell apoptosis, must be activated from the latent form (LTGF-beta) to induce biological responses. In the present study, we report activation of TGF-beta by functional and physical co-operation of the mannose-6-phosphate/insulin-like-growth-factor-II receptor (CD222) and the urokinase-type plasminogen activator receptor (CD87). We show that endothelial cells express CD222 and CD87 in a membrane complex and demonstrate that the association of these two receptors is essential for the release of active TGF-beta in the transduced mouse fibroblast used as model cells.

HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy.

The erythroid phenotype of EKLF-null mice: defects in hemoglobin metabolism and membrane stability.

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression.

Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region.

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, we searched for novel Pax2-regulated genes by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2-/- embryos carrying a Pax2(GFP) BAC transgene. Here, we report the identification of five genes that depend on Pax2 function for their expression in the mid-hindbrain boundary region.

Phosphoproteome and transcriptome analysis of the neuronal response to a CDK5 inhibitor.

In Alzheimer's disease and amyotrophic lateral sclerosis deregulation of cyclin-dependent kinase 5 (CDK5) causes hyperphosphorylation of tau and neurofilament proteins, respectively, leading to neuronal cell death. We have demonstrated recently that pharmacological inhibition of CDK5 protects neurons under various stressful conditions (Weishaupt J. H.

The profile of repeat-associated histone lysine methylation states in the mouse epigenome.

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.

Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes.

Polyomavirus tumorantigens have a profound effect on gene expression in mouse fibroblasts.

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A.

Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.

With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues.

FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1.

Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor-induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes.

Genome-wide analysis of gene expression regulated by the HAT cofactor Trrap in conditional knockout cells.

Transactivation/transformation-domain associated protein (TRRAP) is a component of several multi-protein HAT complexes implicated in both transcriptional regulation and DNA repair. We recently identified Trrap, the murine ortholog of TRRAP, as an essential protein implicated in mitotic progression control, although its target genes are not known. In the present study, we analyzed the expression profiles of Trrap-responsive genes, using cDNA microarray in mitotic cells.

Granulocyte-macrophage colony-stimulating factor-based melanoma cell vaccines immunize syngeneic and allogeneic recipients via host dendritic cells.

Subcutaneous injection of GM-CSF-expressing cancer cells into experimental animals results in protective cancer immunity. To delineate the mode of action of such vaccines, we used trinitrophenyl, the antigenic moiety of the contact allergen trinitrochlorobenzene, as surrogate Ag. Trinitrophenyl-derivatized bone marrow-derived dendritic cells were found to elicit a contact hypersensitivity response in syngeneic, but not in allogeneic recipients, compatible with their expected mode of direct Ag presentation.

Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: molecular analysis by expression profiling.

Erythroid progenitors undergo renewal (proliferation without apparent differentiation) in response to erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids (dexamethasone) (Dex). SCF and Dex cooperate with Epo to promote proliferation and inhibit differentiation of erythroid progenitors, while Epo alone is required to protect erythroid cells from apoptosis during terminal red cell maturation. To examine the mechanism of the synergistic interactions of Epo, SCF, and Dex, we analyzed gene expression patterns using DNA chip-based large-scale comparative gene profiling using microarrays enriched in hematopoietic transcripts or containing randomly selected genes.

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells.

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples.

MLL-ENL cooperates with SCF to transform primary avian multipotent cells.

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF).

DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression.

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes.

Listeria monocytogenes modulates macrophage cytokine responses through STAT serine phosphorylation and the induction of suppressor of cytokine signaling 3.

Macrophage activation as part of natural resistance to infection is caused by stimulation with IFN-gamma and by the invading microorganisms or microbial products. Infection of macrophages with the Gram-positive bacterium Listeria monocytogenes for short periods before activation with IFN-gamma increased the phosphorylation of transcription factor STAT1 at S727 and thereby the expression of IFN-gamma-induced genes. By contrast, persistent infection with viable bacteria or treatment with heat-killed Listeria diminished IFN-gamma-stimulated transcription and the phosphorylation of STAT1 at Y701.

A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry.

In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes.

The glucocorticoid receptor is required for stress erythropoiesis.

The glucocorticoid receptor (GR) coordinates a multitude of physiological responses in vivo. In vitro, glucocorticoids are required for sustained proliferation of erythroid progenitors (ebls). Here, we analyze the impact of the GR on erythropoiesis in vivo, using GR-deficient mice or mice expressing a GR defective for transactivation.

Identification of syntenin as a protein of the apical early endocytic compartment in Madin-Darby canine kidney cells.

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition.

Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma.

The goal of this study was to investigate how bacterial LPS affects macrophage responsiveness to the activating factor IFN-gamma. Pretreatment of macrophages with LPS for <2 h increased the transcriptional response to IFN-gamma. In contrast, simultaneous stimulation with IFN-gamma and LPS, or pretreatment with LPS for >4 h, suppressed Stat1 tyrosine 701 phosphorylation, dimerization, and transcriptional activity in response to IFN-gamma.