bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the brain‐derived neurotrophic factor at glutamatergic synapses
Authors: Carvalho A L , Caldeira M V , Santos S D , Duarte C B 
Published: December 2007

The neurotrophin brain‐derived neurotrophic factor (BDNF) plays an important role in the activity‐dependent regulation of synaptic structure and function, particularly of the glutamatergic synapses. BDNF may be released in the mature form, which activates preferentially TrkB receptors, or as proBDNF, which is coupled to the stimulation of the p75NTR. In the mature form BDNF induces rapid effects on glutamate release, and may induce short‐ and long‐term effects on the postsynaptic response to the neurotransmitter. BDNF may affect glutamate receptor activity by inducing the phosphorylation of the receptor subunits, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. Stimulation of the local protein synthesis and transcription activity account for the delayed effects of BDNF on glutamatergic synaptic strength. Several evidences show impaired synaptic plasticity of glutamatergic synapses in diseases where compromised BDNF function has been observed, such as Huntington's disease, depression, anxiety, and the BDNF polymorphism Val66Met, suggesting that upregulating BDNF‐activated pathways may be therapeutically relevant. This review focuses on recent advances in the understanding of the regulation of the glutamatergic synapse by BDNF, and its implications in synaptic plasticity.British Journal of Pharmacology (2008) 153, S310–S324; doi:10.1038/sj.bjp.0707509; published online 3 December 2007
Paraoxonase 1 gene transfer lowers vascular oxidative stress and improves vasomotor function in apolipoprotein E‐deficient mice with pre‐existing atherosclerosis
Authors: Guns P‐J , Van Assche T , Verreth W , Fransen P , Mackness B , Mackness M , Holvoet P , Bult H 
Published: December 2007

Background and purpose:Transgenesis of human paraoxonase 1 (PON1), a HDL‐associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E‐deficient (apoE−/−) mice with advanced atherosclerosis.Experimental approach:ApoE−/− mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non‐transfected apoE−/− (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta.Key results:PON1 gene transfer raised total PON1 serum activity 13‐15 fold during the 3‐week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque‐free thoracic aorta rings from apoE−/− mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86±2%), ATP (90±2%) or UTP (83±3%). In contrast, in plaque‐bearing segments amplitude (55±7%, 68±8%, 52±8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE−/− SMCs released less intracellular calcium than WT upon sarco‐endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection.Conclusions and implications:These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca2+ homeostasis in segments with pre‐existing atherosclerosis, independently of an effect on plaque size.British Journal of Pharmacology (2008) 153, 508–516; doi:10.1038/sj.bjp.0707585; published online 3 December 2007
Control of left ventricular mass by moxonidine involves reduced DNA synthesis and enhanced DNA fragmentation
Authors: Paquette P‐A , Duguay D , Ayoubi R El‐ , Menaouar A , Danalache B , Gutkowska J , DeBlois D , Mukaddam‐Daher S 
Published: December 2007

Background and purpose:Left ventricular hypertrophy (LVH) is a maladaptive process associated with increased cardiovascular risk. Regression of LVH is associated with reduced complications of hypertension. Moxonidine is an antihypertensive imidazoline compound that reduces blood pressure primarily by central inhibition of sympathetic outflow and by direct actions on the heart to release atrial natriuretic peptide, a vasodilator and an antihypertrophic cardiac hormone. This study investigated the effect of moxonidine on LVH and the mechanisms involved in this effect.Experimental approach:Spontaneously hypertensive rats were treated with several doses of moxonidine (s.c.) over 4 weeks. Blood pressure and heart rate were continuously monitored by telemetry. Body weight and water and food intake were measured weekly. Measurements also included left ventricular mass, DNA content, synthesis, fragmentation, and apoptotic/anti‐apoptotic pathway proteins.Key results:The decrease in mean arterial pressure stabilized at ∼ −10 mm Hg after 1 week of treatment and thereafter. Compared to vehicle‐treated rats (100%), left ventricular mass was dose‐ and time‐dependently reduced by treatment. This reduction remained significantly lower after normalizing to body weight. Moxonidine reduced left ventricular DNA content and inhibited DNA synthesis. DNA fragmentation transiently, but significantly increased at 1 week of moxonidine treatment and was paralleled by elevated active caspase‐3 protein. The highest dose significantly decreased the apoptotic protein Bax and all doses stimulated anti‐apoptotic Bcl‐2 after 4 weeks of treatment.Conclusions and implications:These studies implicate the modulation of cardiac DNA dynamics in the control of left ventricular mass by moxonidine in a rat model of hypertension.British Journal of Pharmacology (2008) 153, 459–467; doi:10.1038/sj.bjp.0707588; published online 3 December 2007
Agonist‐selective mechanisms of GPCR desensitization
Authors: Kelly E , Bailey C P , Henderson G 
Published: December 2007

The widely accepted model of G protein‐coupled receptor (GPCR) regulation describes a system where the agonist‐activated receptors couple to G proteins to induce a cellular response, and are subsequently phosphorylated by a family of kinases called the G protein‐coupled receptor kinases (GRKs). The GRK‐phosphorylated receptor then acts as a substrate for the binding of a family of proteins called arrestins, which uncouple the receptor and G protein so desensitizing the agonist‐induced response. Other kinases, principally the second messenger‐dependent protein kinases, are also known to play a role in the desensitization of many GPCR responses. It is now clear that there are subtle and complex interactions between GRKs and second messenger‐dependent protein kinases in the regulation of GPCR function. Functional selectivity describes the ability of agonists to stabilize different active conformations of the same GPCR. With regard to desensitization, distinct agonist‐activated conformations of a GPCR could undergo different molecular mechanisms of desensitization. An example of this is the μ opioid receptor (MOPr), where the agonists morphine and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) induce desensitization of the MOPr by different mechanisms, largely protein kinase C (PKC)‐ or GRK‐dependent, respectively. This can be best explained by supposing that these two agonists stabilize distinct conformations of the MOPr, which are nevertheless able to couple to the relevant G‐proteins and produce similar responses, yet are sufficiently different to trigger different regulatory processes. There is evidence that other GPCRs also undergo agonist‐selective desensitization, but the full therapeutic consequences of this phenomenon await further detailed study.British Journal of Pharmacology(2008)153, S379–S388; doi:10.1038/sj.bjp.0707604; published online 3 December 2007
Oxytocin stimulates migration and invasion in human endothelial cells
Authors: Cattaneo M G , Chini B , Vicentini L M 
Published: December 2007

Background and purpose:It has recently been reported that oxytocin is produced by some tumour cell types, and that oxytocin receptors, belonging to the G‐protein‐coupled receptor (GPCR) family, are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to oxytocin with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis.Experimental approach:We employed chemotaxis and chemoinvasion assays to characterize the effect of oxytocin on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action.Key results:We showed that oxytocin stimulates migration and invasion in HUVECs via oxytocin receptor activation. Searching for the molecular mechanism(s) responsible for oxytocin's pro‐migratory effect, we identified the Gq coupling of oxytocin receptors and phospholipase C (PLC) as the main effectors of oxytocin's action in HUVECs. We also found that oxytocin stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol‐3‐kinase (PI‐3‐K)/AKT pathway, and that the activation of PI‐3‐K and formation of nitric oxide (NO) are required for the pro‐migratory effect of oxytocin.Conclusions and implications:The ability of oxytocin to stimulate HUVEC motility and invasion suggests that the hormone can participate in physiopathological processes where activation of endothelial cells plays an important role, for example, in angiogenesis. Interestingly, both the AKT and eNOS phosphorylation induced by oxytocin receptor activation depended on PLC activity, thus suggesting the existence of a still undefined mechanism connecting PLC to the PI‐3‐K/AKT pathway, upon oxytocin stimulation. British Journal of Pharmacology (2008) 153, 728–736; doi:10.1038/sj.bjp.0707609; published online 3 December 2007
Region‐specific effects ofN,N′‐dodecane‐1,12‐diyl‐bis‐3‐picolinium dibromide on nicotine‐induced increase in extracellular dopaminein vivo
Authors: Rahman S , Zhang Z , Papke R L , Crooks P A , Dwoskin L P , Bardo M T 
Published: December 2007

Background and purpose:Systemic administration ofN,N′‐dodecane‐1,12‐diyl‐bis‐3‐picolinium dibromide (bPiDDB), an antagonist of nicotinic acetylcholine receptors (nAChRs) attenuated the nicotine‐induced increase in dopamine levels in nucleus accumbens (NAcc).Experimental approach:Usingin vivomicrodialysis, we investigated the effects of local perfusion of the novel nAChR antagonist bPiDDB into the NAcc or ventral tegmental area (VTA) on increased extracellular dopamine in NAcc, induced by systemic nicotine. We also examined the concentration‐dependent effects of bPiDDB on the acetylcholine (ACh)‐evoked response of specific recombinant neuronal nAChR subtypes expressed inXenopusoocytes, using electrophysiological methods.Key results:Nicotine (0.4 mg kg−1, s.c.) increased extracellular dopamine in NAcc, which was attenuated by intra‐VTA perfusion of mecamylamine (100 μM). Intra‐VTA perfusion of bPiDDB (1 and 10 μM) reduced nicotine‐induced increases in extracellular dopamine in NAcc. In contrast, intra‐NAcc perfusion of bPiDDB (1 or 10 μM) failed to alter the nicotine‐induced increase in dopamine in NAcc. Intra‐VTA perfusion of bPiDDB alone did not alter basal dopamine levels, compared to control, nor the increased dopamine in NAcc following amphetamine (0.5 mg kg−1, s.c.). UsingXenopusoocytes, bPiDDB (0.01–100 μM) inhibited the response to ACh on specific combinations of rat neuronal nAChR subunits, with highest potency at α3β4β3 and lowest potency at α6/3β2β3.Conclusions and implications:bPiDDB‐Sensitive nAChRs involved in regulating nicotine‐induced dopamine release are located in the VTA, rather than in the NAcc. As bPiDDB has properties different from the prototypical nAChR antagonist mecamylamine, further development may lead to novel nAChR antagonists for the treatment of tobacco dependence.British Journal of Pharmacology(2008)153, 792–804; doi:10.1038/sj.bjp.0707612; published online 3 December 2007
Testing Spatial and Nonspatial Learning Using a Morris Water Maze
Authors: Paul Carol Ann , Beltz Barbara , Berger-Sweeney Joanne 
Published: December 2007

INTRODUCTIONAnimal studies of memory have used many different types of mazes and tasks, where the animal is required to learn the demands of the test and then perform correctly until a predetermined performance criterion is reached. Traditional studies have relied on water or food deprivation to motivate the animal to do the test using food or water rewards. In 1984, Richard Morris introduced the water maze. The maze consists of a large cylindrical tank of water with a hidden platform; the animal has to swim until it finds the platform. The animal generally uses cues outside the maze (extramaze cues) to develop a spatial map of the environment and guide its performance. This maze avoids the problems associated with food or water deprivation, because the animal’s goal is to find the hidden platform so that it can stop swimming. Many types of memory can be studied by varying the visual cues, the placement of the platform, and the starting point of the animal. This protocol describes the use of the water maze to test rats in two behavioral tasks: a spatial task and a nonspatial, visual task.
Hydroxyl-Radical Footprinting
Authors: Carey Michael , Smale Stephen T. 
Published: December 2007

INTRODUCTIONThe hydroxyl-radical footprinting methodology has wide applications to studying protein-DNA interactions, as well as structural perturbations (e.g., bending) that occur in DNA during protein binding. Hydroxyl radicals cleave DNA by abstracting a hydrogen atom from C4 of the sugar in the minor groove. Protein binding over the minor groove generally protects the sugar from cleavage. Because a hydroxyl radical molecule is small and thus is not subject to the same steric restrictions as other agents (such as DNase I), hydroxyl-radical footprinting can give detailed information on protein binding to the minor groove. In this protocol, the radical is generated by Fe(II) EDTA, which cleaves hydrogen peroxide into a hydroxyl radical and a hydroxide ion. The radical then cleaves the sugar in a diffusion limited reaction. Ascorbic acid is included to regenerate the active Fe(II).
Phosphate Ethylation Interference Assay: Figure 1.
Authors: Carey Michael , Smale Stephen T. 
Published: December 2007

INTRODUCTIONIn the ethylation interference assay, ethyl nitrosourea is used to ethylate phosphates within 32P-end-labeled DNA molecules that contain a protein recognition site; the reaction is optimized to yield approximately one ethyl group per DNA molecule. The DNA is then incubated with barely saturating concentrations of a DNA-binding protein, but the ethylated phosphates interfere with proteins coming into close proximity to the minor groove or phosphate backbone of the binding site on the DNA molecule. Because ethylation makes the DNA susceptible to piperidine-induced cleavage, the protein recognition site can be identified by analyzing the cleavage products (from modified DNA molecules with bound protein versus modified DNA molecules without bound protein) on a polyacrylamide/urea gel alongside a sequencing ladder. Two points are critical for success with this assay. First, only about 10% of the molecules should be ethylated, which avoids having a significant number of molecules with multiple ethylations. This can be determined by modifying the DNA until ~10% of the starting probe is converted to a ladder of bands. It is critical that each DNA molecule be modified only once, so that it is clear that the modification being detected--the one nearest to the 32P-labeled end--is the one responsible for interference. Second, only barely saturating amounts of protein should be bound to the DNA. Barely saturated amounts are used because although the ethylation decreases binding, it does not always abolish it. High concentrations of protein may overcome the deleterious effect of the modification.
Chemical Two-Photon Uncaging
Authors: Pettit Diana L. , Augustine George J. 
Published: December 2007

INTRODUCTIONChemical two-photon uncaging is useful for a wide range of applications, including both mapping of receptor location and localized photostimulation of neurons via activation of excitatory glutamate receptors. Experimental preparations could include brain slices, cultured neurons, and, among other possibilities, whole brains in vivo. This protocol documents the utility of chemical two-photon uncaging in examining glutamate receptors of pyramidal neurons in hippocampal slices.
Ex Ovo Electroporation of DNA Vectors into Pre-gastrulation Avian Embryos
Authors: Cui Cheng , Rongish Brenda , Little Charles , Lansford Rusty 
Published: December 2007

INTRODUCTIONThe transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method allows easier embryo orientation (the posterior half of the embryo is darker than the anterior half). Thus, positioning of the specimen and consistency of the electroporation site between embryos is improved. Furthermore, nearly all embryos can be electroporated at the same stage using the ex ovo method: If some embryos have not reached a desired stage, it is possible to temporally stop development of those embryos already at the desired stage by keeping them at room temperature while incubating the rest at 37°C until they develop. The method described here uses relatively low voltage, and the electroporation chamber can be made easily, with no specialized equipment required.
Preparation of Cells in 96-Well Plates for siRNA Transfection and High-Content Analysis
Authors: Roquemore Elizabeth P. 
Published: December 2007

INTRODUCTIONRNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article details how to perform siRNA transfection in multiwell plates for subsequent high-content analysis.
Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis
Authors: Roquemore Elizabeth P. 
Published: December 2007

INTRODUCTIONRNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article describes how to optimize siRNA knockdown in EGFP-expressing cell lines.
Organotypic Slice Culture of Embryonic Brain Tissue: Figure 1.
Authors: Daza Ray A.M. , Englund Chris , Hevner Robert F. 
Published: December 2007

INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.
Fate-Mapping Technique: Using Carbocyanine Dyes for Vital Labeling of Cells in Gastrula-Stage Mouse Embryos Cultured In Vitro
Authors: Bildsoe Heidi , Franklin Vanessa , Tam Patrick P.L. 
Published: December 2007

INTRODUCTIONThe allocation of different progenitor populations to embryonic structures can be visualized by tracking the distribution of cells to specific tissues in the live embryo. A critical prerequisite for cell tracking is to identify unambiguously the progenitors and their descendants during morphogenesis. This can be achieved by using molecular markers that are expressed from transgenes integrated into the genome or as episomal DNA constructs, or by tagging the cells with exogenous markers that are incorporated into the cell membrane or cytoplasmic components of the cells. These labels can be introduced by dye-labeling the membrane, injecting marker enzyme into the cytoplasm, or integrating reporter constructs by transfection or electroporation. This protocol describes how to label cells in the endoderm (which, at this stage of development, is the superficial tissue layer) of live mouse embryos at 7.0-7.5 days post-coitum (dpc), using two carbocyanine dyes (DiI and DiO).
Vole–vegetation interactions in an experimental, enemy free taiga floor system
Authors: Rammul Üllar , Oksanen Tarja , Oksanen Lauri , Lehtelä Jouko , Virtanen Risto , Olofsson Johan , Strengbom Joachim , Rammul Ivi , Ericson Lars 
Published: December 2007

Vole–vegetation interactions in a predation‐free taiga environment of northern Fennoscandia were studied by transferring vegetation from natural Microtus habitats into a greenhouse, where three habitat islands of about 30 m2 were created. The ‘islands’ were subjected to simulated summer conditions and a paired female field vole, Microtusagrestis, was introduced to each ‘island’. The development of the female and her young was followed by recurrent live trapping. The development of the vegetation was followed by recurrent marking and censusing of plant shoots at intervals of five days. In the next growing season, two ‘islands’ were subjected to a new grazing treatment to study the impacts of repeated grazing on the vegetation and on the growth and reproduction of voles. Plant biomasses were harvested at the end of each trial. In all trials, the biomasses of graminoids and non‐toxic herbs other than ferns, fireweeds and rosaceous plants were profoundly decimated. Even the biomass of a toxic herb Aconitum lycoctonum decreased largely at pace with the palatable herbs. The least preferred plant categories maintained their biomasses at control levels. Their neutral collective response was created by opposite species‐level trends. Species typical for moist and nutrient‐rich forests suffered from vole grazing, whereas the biomass of species adapted to disturbed habitats increased.In spite of the dramatic changes in the vegetation, the introduced female voles survived throughout the trials and reproduced normally. The young of their first litters survived well and reached the final weights typical for individuals starting to winter as immatures. We conclude that most of the plant biomass found on productive boreal forest floors is potential food for field voles and remains palatable for them even when subjected to recurrent, severe grazing. If nothing else than summer resources were limiting the growth of the field vole populations, the plants currently dominating moist and nutrient‐rich taiga floors could not survive in this habitat.