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1.
Background
The question of whether intact somatic cells committed to a specific differentiation fate, can be reprogrammed in vivo by exposing them to a different host microenvironment is a matter of controversy. Many reports on transdifferentiation could be explained by fusion with host cells or reflect intrinsic heterogeneity of the donor cell population.Methodology/Principal Findings
We have tested the capacity of cloned populations of mouse and human muscle progenitor cells, committed to the myogenic pathway, to transdifferentiate to neurons, following their inoculation into the developing brain of newborn mice. Both cell types migrated into various brain regions, and a fraction of them gained a neuronal morphology and expressed neuronal or glial markers. Likewise, inoculated cloned human myogenic cells expressed a human specific neurofilament protein. Brain injected donor cells that expressed a YFP transgene controlled by a neuronal specific promoter, were isolated by FACS. The isolated cells had a wild-type diploid DNA content.Conclusions
These and other results indicate a genuine transdifferentiation phenomenon induced by the host brain microenvironment and not by fusion with host cells. The results may potentially be relevant to the prospect of autologous cell therapy approach for CNS diseases. 相似文献2.
Casey Cook Jennifer Gass Judith Dunmore Jimei Tong Julie Taylor Jason Eriksen Eileen McGowan Jada Lewis Jennifer Johnston Leonard Petrucelli 《PloS one》2009,4(6)
Background
Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function.Methodology/Principal Findings
We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo.Conclusions/Significance
Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s). 相似文献3.
Background
Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Methodology
Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.Conclusions
All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies. 相似文献4.
Adriana M. C. Canavaci Juan M. Bustamante Angel M. Padilla Cecilia M. Perez Brandan Laura J. Simpson Dan Xu Courtney L. Boehlke Rick L. Tarleton 《PLoS neglected tropical diseases》2010,4(7)
Background
The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.Methods
In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC50 lower than that of BZ.Findings
This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.Conclusion
These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo. 相似文献5.
Saederup N Cardona AE Croft K Mizutani M Cotleur AC Tsou CL Ransohoff RM Charo IF 《PloS one》2010,5(10):e13693
Background
Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.Methodology/Principal Findings
We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6Chi/CCR2hi monocytes. Surprisingly, neutrophils, not Ly6Clo monocytes, largely replaced Ly6Chi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.Conclusion/Significance
These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations. 相似文献6.
Background
Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation.Methodology/Principal Findings
From a Drosophila forward genetic screen, we identified a mutation in capulet-encoding a conserved actin-binding protein-that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer''s models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other.Conclusions/Significance
The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer''s and Parkinson''s cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease. 相似文献7.
Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions. 相似文献8.
Lulu Li Jing Sun Changqing Xu Hongying Zhang Jinfeng Wu Baojun Liu Jingcheng Dong 《PloS one》2014,9(8)
Purpose
To investigate the effects of icariin, a major constituent of flavonoids isolated from the herb Epimedium, on cigarette smoke (CS) induced inflammatory responses in vivo and in vitro.Methods
In vivo, BALB/c mice were exposed to smoke of 15 cigarettes for 1 h/day, 6 days/week for 3 months and dosed with icariin (25, 50 and 100 mg/kg) or dexamethasone (1 mg/kg). In vitro, A549 cells were incubated with icariin (10, 50 and 100 µM) followed by treatments with CSE (2.5%).Results
We found that icariin significantly protected pulmonary function and attenuated CS-induced inflammatory response by decreasing inflammatory cells and production of TNF-α, IL-8 and MMP-9 in both the serum and BALF of CS-exposed mice and decreasing production of TNF-α and IL-8 in the supernatant of CSE-exposed A549 cells. Icariin also showed properties in inhibiting the phosphorylation of NF-κB p65 protein and blocking the degradation of IΚB-α protein. Further studies revealed that icariin administration markedly restore CS-reduced GR protein and mRNA expression, which might subsequently contribute to the attenuation of CS-induced respiratory inflammatory response.Conclusion
Together these results suggest that icariin has anti-inflammatory effects in cigarette smoke induced inflammatory models in vivo and in vitro, possibly achieved by suppressing NF-κB activation and modulating GR protein expression. 相似文献9.
Abhiruchi Agarwal Andreas Boettcher Rainer Kneuer Farid Sari-Sarraf Adriana Donovan Julian Woelcke Oliver Simic Trixi Brandl Thomas Krucker 《PloS one》2013,8(2)
Background and Aims
Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis.Methods
Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent “smart” probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging.Results
We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio.Conclusions
We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition. 相似文献10.
Catherine Dostert Greta Guarda Jackeline F. Romero Philippe Menu Olaf Gross Aubry Tardivel Mario-Luca Suva Jean-Christophe Stehle Manfred Kopf Ivan Stamenkovic Giampietro Corradin Jurg Tschopp 《PloS one》2009,4(8)
Background
Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents.Methodology/Principal Findings
We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1β. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K+ efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged.Significance/Conclusions
The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria. 相似文献11.
Background
Synapses exhibit strikingly different forms of plasticity over a wide range of time scales, from milliseconds to hours. Studies on synaptic plasticity typically use constant-frequency stimulation to activate synapses, whereas in vivo activity of neurons is irregular.Methodology/Principal Findings
Using extracellular and whole-cell electrophysiological recordings, we have here studied the synaptic responses at hippocampal mossy fiber synapses in vitro to stimulus patterns obtained from in vivo recordings of place cell firing of dentate gyrus granule cells in behaving rodents. We find that synaptic strength is strongly modulated on short- and long-lasting time scales during the presentation of the natural stimulus trains.Conclusions/Significance
We conclude that dynamic short- and long-term synaptic plasticity at the hippocampal mossy fiber synapse plays a prominent role in normal synaptic function. 相似文献12.
Background
Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).Methods
The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.Results
Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.Conclusions
BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells. 相似文献13.
Background
Previous work has suggested, but not demonstrated directly, a critical role for both glutamatergic and GABAergic neurons of the pontine tegmentum in the regulation of rapid eye movement (REM) sleep.Methodology/Principal Findings
To determine the in vivo roles of these fast-acting neurotransmitters in putative REM pontine circuits, we injected an adeno-associated viral vector expressing Cre recombinase (AAV-Cre) into mice harboring lox-P modified alleles of either the vesicular glutamate transporter 2 (VGLUT2) or vesicular GABA-glycine transporter (VGAT) genes. Our results show that glutamatergic neurons of the sublaterodorsal nucleus (SLD) and glycinergic/GABAergic interneurons of the spinal ventral horn contribute to REM atonia, whereas a separate population of glutamatergic neurons in the caudal laterodorsal tegmental nucleus (cLDT) and SLD are important for REM sleep generation. Our results further suggest that presynaptic GABA release in the cLDT-SLD, ventrolateral periaqueductal gray matter (vlPAG) and lateral pontine tegmentum (LPT) are not critically involved in REM sleep control.Conclusions/Significance
These findings reveal the critical and divergent in vivo role of pontine glutamate and spinal cord GABA/glycine in the regulation of REM sleep and atonia and suggest a possible etiological basis for REM sleep behavior disorder (RBD). 相似文献14.
Objectives
Thrombus and secondary thrombosis plays a key role in stroke. Recent molecular imaging provides in vivo imaging of activated factor XIII (FXIIIa), an important mediator of thrombosis or fibrinolytic resistance. The present study was to investigate the fibrin deposition in a thromboembolic stroke mice model by FXIIIa–targeted near-infrared fluorescence (NIRF) imaging.Materials and Methods
The experimental protocol was approved by our institutional animal use committee. Seventy-six C57B/6J mice were subjected to thromboembolic middle cerebral artery occlusion or sham operation. Mice were either intravenously injected with the FXIIIa-targeted probe or control probe. In vivo and ex vivo NIRF imaging were performed thereafter. Probe distribution was assessed with fluorescence microscopy by spectral imaging and quantification system. MR scans were performed to measure lesion volumes in vivo, which were correlated with histology after animal euthanasia.Results
In vivo significant higher fluorescence intensity over the ischemia-affected hemisphere, compared to the contralateral side, was detected in mice that received FXIIIa-targeted probe, but not in the controlled mice. Significantly NIRF signals showed time-dependent processes from 8 to 96 hours after injection of FXIIIa-targeted probes. Ex vivo NIRF image showed an intense fluorescence within the ischemic territory only in mice injected with FXIIIa-targeted probe. The fluorescence microscopy demonstrated distribution of FXIIIa-targeted probe in the ischemic region and nearby micro-vessels, and FXIIIa-targeted probe signals showed good overlap with immune-fluorescent fibrin staining images. There was a significant correlation between total targeted signal from in vivo or ex vivo NIRF images and lesion volume.Conclusion
Non-invasive detection of fibrin deposition in ischemic mouse brain using NIRF imaging is feasible and this technique may provide an in vivo experimental tool in studying the role of fibrin in stroke. 相似文献15.
Background
Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.Methodology/Principal Findings
Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.Conclusions/Significance
Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins. 相似文献16.
Background
The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.Methodologies/Principal Findings
Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCι) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCι by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).Conclusions/Significance
Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders. 相似文献17.
Shenje LT Field LJ Pritchard CA Guerin CJ Rubart M Soonpaa MH Ang KL Galiñanes M 《PloS one》2008,3(4):e1929
Aims
Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs.Methods
Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies.Results
Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone.Conclusions
This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells. 相似文献18.
Nikolett Lénárt Viktor Szegedi Gábor Juhász Aniko Kasztner János Horváth Erika Bereczki Melinda E. Tóth Botond Penke Miklós Sántha 《PloS one》2012,7(9)
Aims
ApoB-100 is the major protein component of cholesterol- and triglyceride-rich LDL and VLDL lipoproteins in the serum. Previously, we generated and partially described transgenic mice overexpressing the human ApoB-100 protein. Here, we further characterize this transgenic strain in order to reveal a possible link between hypeprlipidemia and neurodegeneration.Methods and Results
We analyzed the serum and cerebral lipid profiles, tau phosphorylation patterns, amyloid plaque-formation, neuronal apoptosis and synaptic plasticity of young (3 month old), adult (6 month old) and aging (10–11 month old) transgenic mice. We show that ApoB-100 transgenic animals present i) elevated serum and cerebral levels of triglycerides and ApoB-100, ii) increased cerebral tau phosphorylation at phosphosites Ser199, Ser199/202, Ser396 and Ser404. Furthermore, we demonstrate, that tau hyperphosphorylation is accompanied by impaired presynaptic function, long-term potentiation and widespread hippocampal neuronal apoptosis.Conclusions
The results presented here indicate that elevated ApoB-100 level and the consequent chronic hypertriglyceridemia may lead to impaired neuronal function and neurodegeneration, possibly via hyperphosphorylation of tau protein. On account of their specific phenotype, ApoB-100 transgenic mice may be considered a versatile model of hyperlipidemia-induced age-related neurodegeneration. 相似文献19.
Background
β2-adrenoceptor agonists elicit bronchodilator responses by binding to β2-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β2-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.Methods
Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β2-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.Results
Temporal analysis revealed that in hASM cells the β2-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.Conclusions
We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β2-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo. 相似文献20.