עבודה זו נתמכה על ידי מכוני הבריאות הלאומיים גרנט GM 69829.

<ol>
	<li>Stephenson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+receptor+theory.">A modification of receptor theory.</a> British Journal of Pharmacology. 11, 379-393 (1956).</li><li>Furchgott, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+b-haloalkylamines+in+the+differentiation+of+receptors+and+in+the+determination+of+dissociation+constants+of+receptor-agonist+complexes.">The use of b-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes.</a> Advances in Drug Research. 3, 21-55 (1966).</li><li>Ehlert, F. J., Suga, H., Griffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+agonism+and+inverse+agonism+in+functional+assays+with+constitutive+activity:+Estimation+of+orthosteric+ligand+affinity+constants+for+active+and+inactive+receptor+states.">Analysis of agonism and inverse agonism in functional assays with constitutive activity: Estimation of orthosteric ligand affinity constants for active and inactive receptor states.</a> J. Pharmacol. Exp. Ther. 33, 671-686 (2011).</li><li>Griffin, M. T., Figueroa, K. W., Liller, S., Ehlert, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+Agonist+Activity+at+G+Protein-Coupled+Receptors:+Analysis+of+M2+Muscarinic+Receptor+Signaling+through+Gi/o,Gs,+and+G15.">Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling through Gi/o,Gs, and G15.</a> J. Pharmacol. Exp. Ther. 321, 1193-1207 (2007).</li><li>Black, J. W., Leff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+models+of+pharmacological+agonism.">Operational models of pharmacological agonism.</a> Proceedings of the Royal Society of LondonSeries B: Biological Sciences. 220, 141-162 (1983).</li><li>Tran, J. A., Chang, A., Matsui, M., Ehlert, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+relative+microscopic+affinity+constants+of+agonists+for+the+active+state+of+the+receptor+in+functional+studies+on+M2+and+M3+muscarinic+receptors.">Estimation of relative microscopic affinity constants of agonists for the active state of the receptor in functional studies on M2 and M3 muscarinic receptors.</a> Mol. Pharmacol. 75, 381-396 (2009).</li><li>Schultz, J., Hamprecht, B., Daly, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+adenosine+3':5'-cyclic+monophosphate+in+clonal+glial+cells:+labeling+of+intracellular+adenine+nucleotides+with+radioactive+adenine.">Accumulation of adenosine 3':5'-cyclic monophosphate in clonal glial cells: labeling of intracellular adenine nucleotides with radioactive adenine.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 1266-1270 (1972).</li><li>Berridge, M. J., Downes, C. P., Hanley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium+amplifies+agonist-dependent+phosphatidylinositol+responses+in+brain+and+salivary+glands.">Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.</a> Biochemical Journal. 206, 587-595 (1982).</li><li>Kendall, D. A., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neurotransmitter Receptor Analysis. , 68-87 (1990).</li><li>Pulido-Rios, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitro+Isolated+Tissue+Functional+Muscarinic+Receptor+Assays.">Vitro Isolated Tissue Functional Muscarinic Receptor Assays.</a> Current Protocols in Pharmacology. 48, 4-15 (2010).</li><li>Kenakin, T., Enna, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Pharmacology. , (2001).</li><li>Ehlert, F. J., Griffin, M. T., Sawyer, G. W., Bailon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+method+for+estimation+of+agonist+activity+at+receptor+subtypes:+comparison+of+native+and+cloned+M3+muscarinic+receptors+in+guinea+pig+ileum+and+transfected+cells.">simple method for estimation of agonist activity at receptor subtypes: comparison of native and cloned M3 muscarinic receptors in guinea pig ileum and transfected cells.</a> Journal of Pharmacology and Experimental Therapeutics. 289, 981-992 (1999).</li><li>Burstein, E. S., Spalding, T. A., Brann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+muscarinic+receptor+subtypes+constitutively+activated+by+G+proteins.">Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins.</a> Mol. Pharmacol. 51, 312-319 (1997).</li></ol>

אין ניגודי אינטרסים הכריז.

בגלל השיטה שלנו להערכת RA אני (יחסית לערך K ב) רק דורש מדידת ריכוז בתגובה עקומות אגוניסט, הניתוח שלנו ניתן לעשות זאת בכל עת עקומות אלה נמדדים. אם יום יומית וריאציה בתגובה של הכנת הניסוי (למשל, תאים או רקמות) הוא גדול, ?...

quantifying agonist activity at g protein-coupled receptors

כאשר אגוניסט מפעיל אוכלוסייה של G-חלבון בשילוב קולטנים (GPCRs), זה מעורר מסלול איתות זה מגיע לשיאו בתגובה של התא או הרקמה. תהליך זה יכול להיות מנותח ברמה של קולטן יחיד, אוכלוסייה של קולטנים, או תגובה במורד הזרם. כאן אנו מתארים כיצד לנתח את התגובה במורד הזרם כדי לקבל אומדן של הזיקה אגוניסט מתמדת המדינה הפעילה של קולטנים יחיד. רצפטורים להתנהג כמו מתגים quantal כי חלופי בין מדינות פעיל פעיל (איור 1). המצב הפעיל אינטראקציה עם חלבונים ספציפיים G או שותפים איתות אחרים. בהעדר ligands, המדינה לא פעיל השולט. הכריכה של אגוניסט מעלה את ההסתברות כי קולטן יעבור למצב פעיל בגלל זיקה מתמדת למדינת פעיל (K ב) שלו הוא גדול בהרבה מזה של מדינת פעיל (K). סיכום שליציאות אקראיות של כל הקולטנים באוכלוסייה התשואות על רמה קבועה של הפעלת קולטן בזמן. הגומלין של ריכוז של אגוניסט לעורר חצי מקסימלי הפעלת קולטן שווה מתמיד את הזיקה שנצפו (K obs), וכן את החלק היחסי של אגוניסט קולטן מתחמי במצב פעיל מוגדר יעילות (ε) (איור 2). שיטות לניתוח התגובות במורד הזרם של GPCRs פותחו המאפשרים הערכה של K obs ואת היעילות היחסית של 1,2 אגוניסט. בדו&quot;ח זה, אנו מראים כיצד לשנות את הניתוח הזה כדי להעריך את ערך K אגוניסט b ביחס לזה של אגוניסט אחר. עבור מבחני כי התערוכה פעילות המכונן, אנו מראים כיצד להעריך K b ביחידות המוחלט של M -1. השיטה שלנו לניתוח ריכוז בתגובה אגוניסט עקומות 3,4 מורכברגרסיה ליניארית הגלובלית באמצעות מודל מבצעי 5. אנו מתארים הליך באמצעות יישום תוכנה, פריזמה (GraphPad תוכנה, Inc, סן דייגו, קליפורניה). ניתוח התשואות אומדן של המכפלה של K ו obs פרמטר יחסי היעילות (τ). אומדן τK obs של אגוניסט אחד, מחולק כי אחרת, היא מדד יחסי של K b (ע&quot;ר i) 6. במשך כל קולטן מפגין פעילות מכוננת, אפשר להעריך פרמטר יחסי היעילות של קומפלקס הקולטן חינם (τ sys). במקרה זה, הערך של K b אגוניסט שווה τK obs / τ sys 3. השיטה שלנו הוא שימושי לקביעת הסלקטיביות של אגוניסט עבור תת קולטן לכימות אגוניסט קולטן חלבוני איתות דרך שונה G.

Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Quantifying Agonist Activity at G Protein-coupled Receptors

שיטה להערכת הזיקה המתמדת של אגוניסט למדינה פעיל (<em&gt; K<subB&gt;</sub</em&gt;) של קולטני G-חלבון בשילוב מתואר. הניתוח מספק אמצעים מוחלט או יחסי של<em&gt; K<subB&gt;</sub</em&gt; תלוי אם הפעלת קולטן המכונן הוא מדיד. השיטה שלנו חל במורד התגובות השונות של הפעלת הקולטן.

כימות פעילות אגוניסט ב G-חלבון יחד רצפטורים

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

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Biology

18.2K Views.  University of California, Irvine. שיטה להערכת הזיקה המתמדת של אגוניסט למדינה פעיל ( K

Video: Quantifying Agonist Activity at G Protein-coupled Receptors

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

שחרור הדופמין ב מסופי Presynaptic הפרט דמיינו עם FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

ב כימות vivo של G מצמידים חלבון קולטן אינטראקציות באמצעות נפתר Spectrally שני פוטון מיקרוסקופית

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

קולטן הדמיה G-חלבון יחד (GPCR) בתיווך אירועים איתות כי הפיקוח של Chemotaxis Discoideum Dictyostelium</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

בדיקות מולקולריות גנטי מקודדות לחקר רצפטורים מצמידים חלבון G

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

אפיון של רצפטורים מצמידים גרמו חלבון על ידי Assay גיוס סיד הקרינה מבוססת

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

חזותי אנדוציטוזה בתיווך clathrin של רצפטורים מצמידים חלבון G בהחלטה חד אירוע באמצעות TIRF מיקרוסקופית

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

התאמת שינויים גנטיים ספציפי DNA מתילציה עם ביטוי ותעתיק פעילות של astrocytic<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

ניתוח השוואתי של הורמון גדילה האנושית בסרום שימוש SPRi, ננו-SPRi ומבחני ELISA

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

G הדמית חלבון בשילוב קולטן בתיווך Chemotaxis ואירועי האיתות שלה בתאי HL60 נויטרופילים דמויים

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

איתור של הפנמת קולטן מצמיד חלבון G-מופעל ליגנד ידי מיקרוסקופי Confocal

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...