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151.
Presynaptic short-term depression is maintained during regulation of transmitter release at a GABAergic synapse in rat hippocampus 总被引:1,自引:0,他引:1
Stefan Hefft Udo Kraushaar Jörg R. P. Geiger Peter Jonas 《The Journal of physiology》2002,539(1):201-208
To examine possible interactions between fast depression and modulation of inhibitory synaptic transmission in the hippocampus, we recorded from pairs of synaptically connected basket cells (BCs) and granule cells (GCs) in the dentate gyrus of rat brain slices at 34 °C. Multiple-pulse depression (MPD) was examined in trains of 5 or 10 inhibitory postsynaptic currents (IPSCs) evoked at frequencies of 10–00 Hz under several conditions that inhibit transmitter release: block of voltage-dependent Ca2+ channels by Cd2+ (10 μ m ), activation of γ-amino-butyric acid type B receptors (GABAB Rs) by baclofen (10 μ m ) and activation of muscarinic acetylcholine receptors (mAchRs) by carbachol (2 μ m ). All manipulations led to a substantial inhibition of synaptic transmission, reducing the amplitude of the first IPSC in the train (IPSC1 ) by 72 %, 61 % and 29 %, respectively. However, MPD was largely preserved under these conditions (0.34 in control versus 0.31, 0.50 and 0.47 in the respective conditions at 50 Hz). Similarly, a theta burst stimulation (TBS) protocol reduced IPSC1 by 54 %, but left MPD unchanged (0.40 in control and 0.39 during TBS). Analysis of both fractions of transmission failures and coefficients of variation (CV) of IPSC peak amplitudes suggested that MPD had a presynaptic expression site, independent of release probability. In conclusion, different types of presynaptic modulation of inhibitory synaptic transmission converge on a reduction of synaptic strength, while short-term dynamics are largely unchanged. 相似文献
152.
Mikko?PS?AresEmail author Maria?Stollenwerk Anneli?Olsson Bengt?Kallin Stefan?Jovinge Jan?Nilsson 《BMC immunology》2002,3(1):13
Background
Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. 相似文献153.
Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier 总被引:6,自引:0,他引:6
Kasten P Vogel J Luginbühl R Niemeyer P Tonak M Lorenz H Helbig L Weiss S Fellenberg J Leo A Simank HG Richter W 《Biomaterials》2005,26(29):5879-5889
Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks. 相似文献
154.
Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix 总被引:3,自引:0,他引:3
The purpose of this study was to evaluate the morphologic and biochemical behavior and activity of human chondrocytes taken from nonarthritic and osteoarthritic cartilage and seeded on a three-dimensional matrix consisting of collagen types I, II, and III. Human articular chondrocytes were isolated from either nonarthritic or osteoarthritic cartilage of elderly subjects, and from nonarthritic cartilage of an adolescent subject, seeded on collagen matrices, and cultured for 12 h, 7 days, and 14 days. Histological analysis, immunohistochemistry, and biochemical assays for glycosaminoglycans (GAGs) and DNA content were performed for cell-seeded and unseeded matrices. Chondrocytes of nonarthritic cartilage revealed a larger number of spherical cells, consistent with a chondrocytic phenotype. The biochemical assay showed a net increase in GAG content in nonarthritic chondrocytes, whereas almost no GAGs were seen in osteoarthritic cells. The DNA results suggest that more osteoarthritic cells than chondrocytes from nonarthritic cartilage attached to the matrix within the first week. Human articular chondrocytes isolated from osteoarthritic cartilage seem to have less bioactivity after expansion and culture in a sponge consisting of type I, II, and III collagen compared with chondrocytes from nonarthritic cartilage. 相似文献
155.
Diffuse leiomyomatosis is associated with the inherited kidney disease Alport syndrome, and characterized by visceral smooth muscle overgrowth within the respiratory, gastrointestinal and female reproductive tracts. Although partial deletions of the type IV collagen genes COL4A5 and COL4A6, paired head-to-head on chromosome Xq22, are known to cause diffuse leiomyomatosis, loss of function for type IV collagen does not explain smooth muscle overgrowth. To further clarify pathogenic mechanisms, we have characterized novel deletions in patients with Alport syndrome-diffuse leiomyomatosis or Alport syndrome alone. A 27.6-kb deletion, in a female with Alport syndrome-diffuse leiomyomatosis, is marked by the most proximal, i.e. most 5', COL4A5 breakpoint described to date. By comparing this deletion to others described here and previously, we have defined a minimal overlap region, only 4.2 kb in length and containing the COL4A5-COL4A6 proximal promoters, loss of which contributes to smooth muscle overgrowth. A novel deletion in a male with Alport syndrome alone is>1.4 Mb in length, encompassing COL4A5 and COL4A6 entirely, as well as neighboring genes. We postulate that loss of the 4.2-kb region in diffuse leiomyomatosis causes misregulation of neighboring genes, contributing to smooth muscle overgrowth. Deletion of the neighboring genes themselves may afford protection from this condition. 相似文献
156.
Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue. 总被引:2,自引:0,他引:2
Stefan Jüttner Michael Vieth Stephan Miehlke Wulf Schneider-Brachert Christian Kirsch Thilo Pfeuffer Norbert Lehn Manfred Stolte 《Modern pathology》2004,17(6):684-689
Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy. 相似文献
157.
The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.Currently available mass-spectrometry methods (Rix and Superti-Furga 2009; Martinez Molina et al. 2013; Savitski et al. 2014; Huber et al. 2015; Drewes and Knapp 2018) for monitoring the effects of cellular perturbations on proteomes cannot be scaled efficiently to monitor time-dependent effects in high throughput. A different approach to study drug action is live-cell imaging of protein dynamics in cells expressing a protein of interest fused to a fluorescent tag. Traditionally, such reporter cells are generated either by overexpression to nonphysiologic levels, by oligonucleotide-directed homologous recombination in yeast, or by using CRISPR-Cas9 and homology-directed repair (HDR) to endogenously tag proteins in human cells (Ghaemmaghami et al. 2003; Huh et al. 2003; Chong et al. 2015; Leonetti et al. 2016). In addition to those targeted approaches, “gene trapping” or “CD-tagging” strategies, which rely on the random, viral integration of fluorescent tags as synthetic exons, have been used for analyzing dynamic changes in response to drugs (Jarvik et al. 1996; Morin et al. 2001; Cohen et al. 2008; Kang et al. 2016), but they are limited by integration site biases and require the isolation and characterization of clones before using them in an arrayed format. Recently, a strategy combining genome engineering and gene trapping using homology-independent CRISPR-Cas9 editing to place a fluorescent tag as a synthetic exon into introns of individual target genes has been described (Serebrenik et al. 2019). The strategy relies on a generic sgRNA excising a fluorescent tag flanked by splice acceptor and donor sites from a generic donor plasmid, which is coexpressed with a gene-specific intron-targeting sgRNA specifying the integration site. Here we show the scalability of that strategy to enable pooled protein tagging of more than 900 metabolic enzymes and epigenetic modifiers. Exposing the GFP-tagged cells to compounds allows us to monitor drug effects on the localization and levels of hundreds of proteins in real time in a pooled format, followed by identification of responding clones by in situ sequencing of the expressed intron-targeting sgRNA that corresponds to the tagged protein (Fig. 1A).Open in a separate windowFigure 1.Pooled GFP intron-tagging of metabolic enzymes. (A) Schematic outline of the approach. (B) Identification of targetable introns within metabolic genes. (C) FACS sorting of clones with successful GFP-tagging by signal enrichment over background mCherry intensity used as control for autofluorescence. (D) Representative image of sorted GFP-tagged cell pool. Scale bar, 25 µm. (E) Comparison of RNA-seq expression in HAP1 cells between genes for which GFP-tagged cells could be isolated and genes that were targeted in the sgRNA library but did not result in successful clone isolation. 相似文献
158.
Hunziker L Recher M Macpherson AJ Ciurea A Freigang S Hengartner H Zinkernagel RM 《Nature immunology》2003,4(4):343-349
Polyclonal hypergammaglobulinemia is a characteristic of chronic inflammatory conditions, including persisting viral infections and autoimmune diseases. Here we have studied hypergammaglobulinemia in mice infected with lymphocytic choriomeningitis virus (LCMV), which induces nonspecific immunoglobulins as a result of switching natural IgM specificities to IgG. The process is dependent on help from CD4+ T cells that specifically recognize LCMV peptides presented by B cells on major histocompatibility complex class II molecules. Thus, hypergammaglobulinemia may arise when specific helper T cells recognize B cells that have processed viral antigens irrespective of the B cell receptor specificity. This nonspecific B cell activation may contribute to antibody-mediated autoimmunity. 相似文献
159.
Analysis of gene expression patterns in small amounts of human ventricular myocardium by a multiplex RNase protection assay 总被引:3,自引:0,他引:3
C. Mittmann Ursula Münstermann Joachim Weil Michael Böhm Stefan Herzig Christoph Nienaber Thomas Eschenhagen 《Journal of molecular medicine (Berlin, Germany)》1998,76(2):133-140
End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These
changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology,
clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the
quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the
RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium
comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts
with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the β1-adrenoceptor, the stimulatory G protein α-subunit (Gsα), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), β-myosin heavy chain (β-MHC), and the atrial natriuretic
peptide (ANP). We extracted 10.7±2.1 μg total RNA from three myocardial biopsies taken in vitro. All of the six genes were
measurable in duplicate in a total of 7 μg RNA. mRNAs of β1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively,
whereas β-MHC and Gsα mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific
and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsα levels obtained by Northern blot analysis with 7.5 μg total RNA showed a good correlation between the two methods. The RNase
protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies.
Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial
ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.
Received: 12 May 1997 / Accepted: 8 September 1997 相似文献
160.