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Suggestion: Table B-1: Character formatting Purpose Shortcut Bold type CTRL+B Italics CTRL+I Changing upper and lower case of letters SHIFT+F3 Subscribing text (automatic spacing) CTRL+EQUAL SIGN Superscribing text (automatic spacing) CTRL+PLUS SIGN Back to standard text CTRL+SHIFT+Z Frames Frames are ideal for summarising a chapter or giving instructions order extra super levitra 100 mg overnight delivery fluoride causes erectile dysfunction. Working with Word Planning a medical textbook ƒ Only write if you want your book to be No order extra super levitra 100mg without a prescription erectile dysfunction age 50. Those who cannot perform this task themselves should delegate the job to a professional reader 100mg extra super levitra amex impotence quoad hoc meaning. Keyboard shortcuts You write the text with your fingers, so you should use the many keyboard shortcuts. Your hand then stays on the keyboard, and you save yourself the trouble of reaching for the mouse. A detailed survey of keyboard combinations can be found in Tables B-1 to B-14. More detailed lists are available on the internet at http://hiv. You should use the following shortcuts: ƒ ALT+CTRL+N: Switch to normal view ƒ ALT+CTRL+I: Switch in or out of print view ƒ ALT+CTRL+O: Switch to outline view ƒ ALT+R: Switch to reading view ƒ CTRL+N: The paragraph where the cursor is located is given the template “Normal” (body text) ƒ ALT+CTRL+1: Paragraph becomes “Heading 1”, the first level of subdivision ƒ ALT+CTRL+2: Paragraph becomes “Heading 2”, the second level of subdivision ƒ ALT+CTRL+3: Paragraph becomes “Heading 3”, the third level of subdivision ƒ SHIFT+ALT+ARROW UP or DOWN: Shifts paragraphs or lines in a table upwards or downwards 85 Materials Table B-2: Windows Purpose Shortcut Closes the window CTRL+F4 Changes to next window CTRL+F6 Changes to previous window CTRL+SHIFT+F6 Maximises window CTRL+F10 Table B-3: Formatting paragraphs(1) Orientation and indents Shortcut Centring a paragraph CTRL+E Full justification of a paragraph CTRL+J Justified left orientation of a paragraph CTRL+L Justified right orientation of a paragraph CTRL+R Creating a hanging indent CTRL+T Removing a hanging indent CTRL+SHIFT+T Removal of paragraph formatting CTRL+Q Table B-4: Formatting paragraph (2) Allocation of templates Shortcut Allocation of the template Standard CTRL+SHIFT+N Allocation of the template Heading 1 ALT+CTRL+1 Allocation of the template Heading 2 ALT+CTRL+2 Allocation of the template Heading 3 ALT+CTRL+3 Allocation of the template Bullets CTRL+SHIFT+L 86 B. Working with Word Table B-5: Copying and shifting texts and diagrams using shortcuts Purpose Shortcut Copying texts or diagrams CTRL+C Pasting texts or diagrams CTRL+V Copying formatting CTRL+SHIFT+C Pasting formatting CTRL+SHIFT+V Table B-6: Deleting texts and diagrams using shortcuts Purpose Shortcut Deleting a word to the left of the cursor CTRL+BACKSPACE Deleting a word to the right of the cursor CTRL+DELETE Cutting highlighted text and filing it on the clipboard CTRL+X Undoing the last action CTRL+Z Cutting and filing in the collection CTRL+F3 Pasting contents of collection CTRL+SHIFT+F3 Table B-7: Pasting special characters Purpose Shortcut Page break CTRL+ENTER Create a nonbreaking hyphen CTRL+ HYPHEN (-) Hard hyphen CTRL+_ Insert a nonbreaking space CTRL+SHIFT+ SPACE Copyright symbol: © ALT+CTRL+C Symbol for a registered trademark: ® ALT+CTRL+R Trademark symbol: ™ ALT+CTRL+T Ellipsis ALT+CTRL+FULL STOP (. Working with Word Table B-11: Editing text in outline view Upgrading, downgrading and shifting Shortcut paragraphs Upgrading a paragraph ALT+SHIFT+ LEFT ARROW Downgrading a paragraph ALT+SHIFT+ RIGHT ARROW Changing into text body CTRL+N Shifting the highlighted paragraph up ALT+SHIFT+UP Shifting the highlighted paragraph down ALT+SHIFT+DOWN Table B-12: Changing the display in outline view Purpose Shortcut Expanding text under a heading ALT+SHIFT+PLUS Reducing text under a heading ALT+SHIFT+MINUS Expand or collapse all text or headings ALT+SHIFT+A Showing the first line or the whole body of text ALT+SHIFT+L Showing all headings on level 1 ALT+SHIFT+1 Showing all headings down to level n ALT+SHIFT+n Table B-13: Working in windows and dialogue windows Switching between windows Shortcut Next application ALT+TAB Previous application ALT+SHIFT+TAB 89 Materials Table B-14: Function key shortcuts Function key SHIFT CTRL CTRL+ SHIFT F3 Change the Cut and file in Paste collection upper and lower collection contents case of letters F4 Repeat Close document instruction search or go-to F5 Go-to Return to Restore Editing a text (Menu Edit) previous previously marker working position shown size of a document window F6 Move on to next Return to document previous window document window F7 Spell Check Thesaurus (Menu Tools) (Menu Tools) F9 Update selected Show field Insert an empty Undo linkage of fields function/field field field finding 90 C. Copyright Removal = your mother language HIV Medicine Free Book Initiative HIV Medicine 2005 is a medical textbook that provides a comprehensive and up-to-date overview of the treatment of HIV Infection (800 pages, ISBN 3- 924774-44-7). Under certain conditions, the editors and the authors of HIV Medicine 2005 agree to remove the copyright on their book for all languages except English and . You could therefore translate the content of HIV Medicine 2005 into any language except and publish it under your own name. This policy is in accordance with the Amedeo Free Book Initiative. To benefit from this offer, you have to comply with the following conditions: 1. Reproduction of the content of this site is not permitted in English or in . You may apply for translation into no more than two languages. You may publish the translation under your own name. However, the main page of the publication – be it the home page of a website or a book cover – must mention the source of the information in this way: Adapted from www. In addition, the authors of the individual chapters have to be mentioned at the beginning of every single chapter. The translation into any other language must reproduce the original documents faithfully. However, if national treatment guidelines, drug approval conditions or treatment-related issues specific to your country differ from what is recommended or 91 Materials described in HIV Medicine 2005, you must add a note to point out that difference. Neither the Publisher nor the editors of HIV Medicine 2005 assume any responsibility for the quality of your translation or for possible injuries and/or damages to persons or property caused by the use of your translation. Pay the greatest attention when translating crucial information such as dosage, dosage schedules, therapeutic regimens, drug descriptions, etc. Before publishing the translation of HIV Medicine 2005, include a disclaimer statement. Translating the text into any language does not confer on you any exclusive rights for that given language. If other working groups wish to translate HIV Medicine 2005 into the same language as you do, we encourage them to do so. Under no circumstances may a translated version be re-translated into English or (see above). Please note that when submitting your data, you may indicate that you wish to be put in contact with other people who intend to translate HIV Medicine 2005 into the same language as you do. To apply for permission to translate HIV Medicine 2005, please submit your name, affiliation, e-mail address and phone number. Usually, your request will be processed within less than 72 hours. You need our written consent to proceed with the translation. A doctor who publishes his own textbooks can earn many times what he would be paid in royalties by a publishing house. More important than this, however, is the fact that a doctor who writes and publishes wants his texts to be read by as many colleagues, students and patients as possible. Te best way to achieve this is through free parallel publication of these texts on the internet. Free Medical Information describes how to produce a successful medical textbook: from defining the project, selecting the co- authors and fixing the deadlines to building the website, printing, marketing, distributing, and negotiating with the sponsors. A book for future publishers and authors, for doctors and students Free – for all those who would like to know how medical textbooks are produced today. Medical Bernd Sebastian Kamps (BSK) is the director of the international Amedeo Literature Project (www. Yanowitz, MD Professor of Medicine University of Utah School of Medicine Medical Director, Intermountain Healthcare ECG Services LDS Hospital & Intermountain Medical Center Salt Lake City, Utah frank. Lindsay, MD (1923-1987) master teacher of electrocardiography, friend, mentor, and colleague. Many of the excellent ECG tracings illustrated in this learning program are from Dr. The materials presented in the “Introduction to ECG Interpretation” Booklet are for your information only. You accept all risk of use of, and reliance on, the materials contained in the Booklet. This document and the ECG website offer an introduction to clinical electrocardiography. ECG terminology and diagnostic criteria often vary from book to book and from one teacher to another. In this document an attempt has been made to conform to standardized terminology and criteria, although new diagnostic concepts derived from the recent ECG literature have been included in some of the sections. The sections in this booklet are organized in the same order as the recommended step- wise approach to ECG interpretation outlined in Section 2 (p7). Beginning students should first go through the sections in the order in which they are presented. Others may choose to explore topics of interest in any order they wish. It is hoped that all students will be left with some of the love of electrocardiography shared by Dr. This list (updated in 2016) is provided on the following page and is also found on http://ecg. Students of electrocardiography are encouraged to study this list and become familiar with the ECG recognition of these diagnoses.

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Manios EG purchase extra super levitra 100mg without prescription erectile dysfunction lisinopril, Mavrakis HE order 100 mg extra super levitra fast delivery erectile dysfunction devices, Kanoupakis EM buy discount extra super levitra 100 mg erectile dysfunction tulsa, Randomized comparison of anterolateral et al. Effects of amiodarone and diltiazem on versus anteroposterior electrode position for persistent atrial fibrillation conversion and biphasic external cardioversion of atrial recurrence rates: a randomized controlled fibrillation. Impact of biphasic electrical cardioversion Efficacy and impact of monophasic versus of atrial fibrillation on early recurrent atrial biphasic countershocks for transthoracic fibrillation and shock efficacy. J Cardiovasc cardioversion of persistent atrial fibrillation. Effects of diltiazem pretreatment on Is pretreatment with ibutilide useful for direct-current cardioversion in patients with atrial fibrillation cardioversion when persistent atrial fibrillation: a single-blind, combined with biphasic shock? Effect of verapamil on secondary Biphasic versus monophasic shock for cardioversion in patients with early atrial external cardioversion of atrial flutter: a fibrillation recurrence after electrical prospective, randomized trial. VERDICT: the Verapamil versus transthoracic atrial defibrillation. Am Heart Digoxin Cardioversion Trial: A randomized J. Efficacy of transthoracic cardioversion of J Cardiovasc Electrophysiol. Randomized study comparing duty-cycled bipolar and unipolar radiofrequency with 199. External undergoing mitral valve surgery: the cardioversion of atrial fibrillation: SWEDish Multicentre Atrial Fibrillation comparison of biphasic vs monophasic study (SWEDMAF). Atrial fibrillation catheter ablation versus Comparison of cool tip versus 8-mm tip surgical ablation treatment (FAST): a 2- catheter in achieving electrical isolation of center randomized clinical trial. PMID: Left atrial ablation versus biatrial ablation 18242535. Left atrial radiofrequency ablation during 16278360. Ablation for longstanding permanent atrial PMID: 19944393. PMID: Does electrogram guided substrate ablation 19084800. A prospective, randomized Pulmonary vein isolation using segmental study. J Interv Card Efficacy of an additional MAZE procedure Electrophysiol. PMID: using cooled-tip radiofrequency ablation in 18418704. Catheter ablation of atrial fibrillation in 2002;23(7):558-66. J Cardiovasc paroxysmal patients: randomized Electrophysiol. Circ Arrhythm fractionated atrial electrograms versus Electrophysiol. PMID: potential-guided pulmonary vein antrum 22139886. A short-term, randomized, double-blind, Catheter ablation treatment in patients with parallel-group study to evaluate the efficacy drug-refractory atrial fibrillation: a and safety of dronedarone versus prospective, multi-centre, randomized, amiodarone in patients with persistent atrial controlled study (Catheter Ablation For The fibrillation: the DIONYSOS study. Left atrial posterior wall isolation does not Circumferential pulmonary-vein ablation for improve the outcome of circumferential chronic atrial fibrillation. J Am radiofrequency catheter ablation in patients Coll Cardiol. Van Breugel HN, Nieman FH, Accord RE, during atrial fibrillation ablation in patients et al. A prospective randomized multicenter without atrial flutter: a randomised comparison on health-related quality of life: controlled trial. J Comparison of antiarrhythmic drug therapy Cardiovasc Electrophysiol. Substrate and Trigger Ablation for Reduction of Atrial Fibrillation (STAR AF): 229. Mitral valve surgery plus concomitant Amiodarone to prevent recurrence of atrial atrial fibrillation ablation is superior to fibrillation. Canadian Trial of Atrial mitral valve surgery alone with an intensive Fibrillation Investigators. Prospective Efficacy of three different ablative randomized comparison of left atrial and procedures to treat atrial fibrillation in biatrial radiofrequency ablation in the patients with valvular heart disease: a treatment of atrial fibrillation. Substrate modification combined with Impact of systematic isolation of superior pulmonary vein isolation improves outcome vena cava in addition to pulmonary vein of catheter ablation in patients with antrum isolation on the outcome of persistent atrial fibrillation: a prospective paroxysmal, persistent, and permanent atrial randomized comparison. Effectiveness of the maze procedure using cooled-tip radiofrequency ablation in 248. Maintenance of sinus rhythm in patients with atrial fibrillation: an 249. De Simone A, De Pasquale M, De Matteis AFFIRM substudy of the first C, et al. Combined radiofrequency modified maze and mitral valve procedure through a port 250. Isolation: long-term results of a prospective PMID: 12895612. Randomized study of surgical isolation of the pulmonary veins for correction of 251. J Thorac Cardiovasc ablation strategies in patients with Surg. Results from a prospective randomized Pulmonary venous isolation versus study. Long-term efficacy and safety of propafenone and sotalol for the maintenance 253. Jessurun ER, van Hemel NM, Defauw JJ, et Randomized comparison between al. A randomized study of combining maze pulmonary vein antral isolation versus surgery for atrial fibrillation with mitral complex fractionated electrogram ablation valve surgery. Ablation of superior pulmonary of catheter ablation and surgical CryoMaze veins compared to ablation of all four procedure in patients with long-lasting pulmonary veins. J Cardiovasc persistent atrial fibrillation and rheumatic Electrophysiol. Comparison of effectiveness of circumferential pulmonary vein isolation carvedilol versus bisoprolol for maintenance preferable to stepwise segmental pulmonary of sinus rhythm after cardioversion of vein isolation for patients with paroxysmal persistent atrial fibrillation. J isolation for atrial fibrillation: a randomized Cardiovasc Electrophysiol. Kochiadakis GE, Igoumenidis NE, Hamilos Noninducibility of atrial fibrillation as an MI, et al. Long-term maintenance of normal end point of left atrial circumferential sinus rhythm in patients with current ablation for paroxysmal atrial fibrillation: a symptomatic atrial fibrillation: amiodarone randomized study.

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Along with inferior frontal gyrus (IFG) and inferior parietal lobe (IPL) buy 100mg extra super levitra mastercard erectile dysfunction johns hopkins, the posterior superior temporal sulcus (STS) is an important component of the MNS cheap extra super levitra 100 mg online erectile dysfunction treatment center. This region contains cells which activate when actions are observed purchase extra super levitra 100 mg without a prescription erectile dysfunction doctors staten island, but not when activation is executed. Gallagher and Frith (2003) proposed that initial analysis of social cues occurs in this region. When a movement is observed there is a progression of neural activity across the brain in the following order (Nishitani et al, 2002). Further, brain areas involved in emotional process (such as the anterior insula and cingulate cortex) are activated when we observe the emotions of others. And, it is proposed that “motor simulation may be a trigger for the simulation of associated feeling states” (Bastiannsen et al, 2009). While discovery of the MN and the MNS has been exciting, some reservations have been expressed (Pascolo et al, 2010). It is probable that this phenomenon also contributes (along with the MNS and other potential systems) to ToM skills. ToM in clinical disorders ToM skills are recently evolved and finely tuned, and depend on a high degree of biological, psychological and sociological integration. Finely balanced equipment is easily disrupted, and it would be reasonable to expect that some psychiatric disorders are underpinned by disrupted ToM processes (Brune et al, 2003; Brune and Brune- Cohrs, 2006). Developmental disorders Children with autism have difficulties with emotional relationships and language acquisition. They have markedly impaired ToM skills (Baron-Cohen, 1988). With maturation, in the teen years, the performance of people with autism on ToM tests, may improve. Interestingly, the lack of ToM skills means these children are unable to deceive others, or to recognize when they are being deceived by others. People with less severe autistic-spectrum disorders have less impairment of ToM skills. There have been conflicting reports about whether MN dysfunction is (Perkins et al, 2010) or is not (Fan et al, 2010) a feature of autism. However, there is no doubt that ToM is impaired in autism spectrum disorders (ASD; Schneider et al, 2013). Personality disorder People with psychopathy are described as being cold and lacking in empathy. This suggests they would perform poorly on ToM tasks. Contrary to expectations, however, people with psychopathy appear to have unimpaired ToM skills (Blair et al, 1996). It is believed that people with psychopathy are aware of the distress they cause others, but are not distressed by these facts. Delusional disorder (DD) ToM is believed to have roots in the evolutionary process, and the “pure” delusions of DD (fear of gangs, jealousy, erotomania and somatic) have been proposed as having had survival value. These delusions, which arise exclusively in a social context, have been labelled, “Theory of Mind Delusions” (Charlton and McClelland, 1999; Charlton, 2003). However, people suffering DD may perform normally on ToM tests (Walston et al, 2000; Bommer and Brune, 2006). This may suggest that current ToM tests are not valid. Alternatively, there may be an explanation in differences between the DD delusions and the “bizarre” delusions of other psychoses. With respect to delusions in general, not specifically those of DD, Freeman (2007) found that ToM deficits may be present, but “they are certainly not specific or necessary”. With respect to delusions in schizophrenia, Pousa et al (2008) reported, “specific ToM deficits were found associated with delusions”. Schizophrenia Frith (1992) raised the possibility of ToM deficits underpinning schizophrenia. He offered a comprehensive theory, with different types of ToM skills impairment accounting for the different symptom groups: positive, negative and disorganization symptoms. Spong et al, (2007) conducted a meta-analysis of studies in schizophrenia and found a highly significant (P < 0. Bora et al (2009) conducted a meta-analysis of 36 studies. In remitted schizophrenia patients the degree of ToM impairment was less pronounced than non-remitted patients, but was still significant. They found evidence of a trait relating to ToM impairment. This is consistent with the finding of Dworkin et al (1993) of impaired social functioning (but not specifically ToM) in the relatives of people with schizophrenia. Bora and Pantelis (2013) found that ToM was substantially impaired at the first episode of schizophrenia, and the deficits were comparable with findings in chronic patients. They also found measurable deficits in ultra-high risk individuals and unaffected relatives (replicated by Cella et al, 2015 and Ho et al, 2015). Other components of social cognition and cognitive deficits are surely involved in schizophrenia, but the evidence indicates a role for ToM deficits in at least some individuals (Langdon et al, 2008). Gavilan Ibanez and Garcia-Albea (2013) report, “In schizophrenia, the deficit in ToM appears to be specific and not dependent on more general cognitive abilities…” Brune (2005) made the observation that people with schizophrenia, in contrast to people with personality disorder, rarely cheat or manipulate others (including their therapists). This makes theoretical sense: with impaired ability to understand the mind of others, the capacity to manipulate the minds of others would be reduced. The other side of this coin fits with the clinical observation that it can be difficult to establish an empathic relationship with some people with schizophrenia. A recent study of patients with schizophrenia demonstrated ToM deficits were positively correlated with grey matter reductions in the STS and medial prefrontal cortex (Koelkebeck et al, 2013). These deficits are not related with performance on executive functioning (Bora, et al 2015) ToM tests ToM testing commenced in the study of autism. There have been advances, but standardised techniques have not been widely agreed. Normal children will pass this test by 3-4 years of age. Children with autism may not pass such tests; if they do so, it is usually at an older age than those without autism. Normal children will pass such tests at around 6 years of age. High functioning children with autism may also pass these tests, but not before their teen years. Other tests include the ability to understand metaphor, sarcasm, humour and faux pas (breach of etiquette; Baron-Cohen, 2001). Theory of Mind Picture Stories Task Dr Martin Brune of University of Bochum, Germany, (Martin.

By compar- There are many ways of detecting phosphorylated proteins generic extra super levitra 100 mg free shipping sublingual erectile dysfunction pills. Subsequently purchase 100mg extra super levitra free shipping erectile dysfunction cure video, these fragments can be isolated and fur- PAGE best extra super levitra 100 mg erectile dysfunction doctors huntsville al, proteins dissolved in SDS are loaded onto one end ther analyzed by methods such as Edman degradation or of a porous gel and exposed to an electric field, which causes mass spectrometry. By using Methods to Examine Regulation of appropriate radiolabeled compounds (such as inorganic Receptors byLocalization and Trafficking phosphate added to the culture medium), it is possible to apply the technique of autoradiography to specifically detect It has been appreciated for many years that a critical parame- radioactive, phosphorylated proteins resolved by SDS- ter that can regulate the strength of functional signal trans- PAGE. It is also possible to use gel electrophoresis to sepa- duction via GPCRs is the actual number of receptors present rate proteins according to relative charge, a property that in target tissues and, in particular, the number of receptors is modified predictably by certain modifications such as present in the plasma membrane of individual cells. These types of separation can be com- disturbances in the regulation of receptor number and/or bined in the use of two-dimensional gel electrophoresis, distribution may be of primary importance in the patho- 22: G-Protein–Coupled Receptors 283 physiology of certain neuropsychiatric disorders. For exam- which antibodies recognizing the native receptor are not ple, long-term administration of dopamine receptor antago- available. In either case the general scheme is as follows: nists can induce upregulation of specific receptors, which Cells or tissues expressing the receptor of interest are fixed may contribute to the apparent supersensitivity of dopamine using standard histologic methods. The fixed cells or tissue receptors associated with tardive dyskinesia (46). This phenomenon is termed bodies recognizing the receptor of interest. Studies using ra- tive epitopes in the specimen (typically several hours), the dioligand binding and subcellular fractionation techniques specimens are washed extensively to remove nonspecifically provided early evidence that multiple mechanisms are capa- associated antibodies. The directly visualizing the subcellular localization of GPCRs secondary antibody is typically coupled to a fluorochrome and for performing biochemical studies of specific receptor (such as fluorescein), a recognizable particle (such as colloi- trafficking mechanisms. GPCRs can be detected in situ in cell or tissue preparations using immunochemical techniques and receptor-specific an- Biochemical Methods to AssaySpecific tibodies. Antibodies that recognize the native receptor pro- Receptor Trafficking Processes tein can be used to examine the localization of endogenously expressed receptors, whereas epitope-tagging methods (see Whereas microscopic imaging can readily provide a great above) can be used to detect mutated versions of the receptor deal of qualitative information about GPCR localization protein or as a means to detect recombinant receptors for and trafficking, it can be quite challenging to quantitiate A B FIGURE 22. Visualization of HA epitope-tagged dopamine D1 receptors in transfected cells, using a fluorochrome-labeled secondary antibody and fluorescence microscopy. The ability of this receptortoundergoregulatedinternalizationis indicatedbythedopamine-inducedredistribution of immunoreactive receptors from the plasma membrane (visualized as linear staining at the cell periphery) to endocytic vesicles (visualized as punctate structures located throughout the cytoplasm). In addition to being extremely a specific subcellular localization or to measure accurately useful for examining posttranslational modifications of the rate or extent of specific trafficking processes. The im- GPCRs, in some cases it is possible to use these techniques portance of these processes has motivated the development to isolate receptor-containing complexes that presumably of biochemical methods for examining GPCR trafficking. The In addition to their utility for receptor localization, antibod- basic idea is to immunopurify a specific GPCR from cell ies specifically recognizing GPCRs facilitate biochemical or tissue extracts (or from a partially purified subcellular studies of GPCR trafficking using techniques adapted from fraction prepared from a cell or tissue lysate) using an anti- other areas of cell and molecular biology. For example, one body recognizing the native receptor or an engineered epi- method that has been extremely useful for quantitative stud- tope tag, and then to analyze proteins specifically associated ies of GPCR endocytosis is cell-surface biotinylation cou- with this complex using a different antibody. In general, pled with immunoprecipitation of receptors. Proteins pres- this is accomplished by immunoprecipitation of the receptor ent in the plasma membrane of cells can be specifically followed by analysis of associated proteins in the complex labeled by incubating intact cells in the presence of biotin by immunoblotting with the appropriate additional anti- coupled to an activated ester, which is membrane-imper- body. In some cases, the protein complexes are sufficiently meant and therefore forms a covalent bond only with ex- stable that they remain associated through the initial immu- posed amine moieties present in plasma membrane proteins. In other cases this is not true, In general, biotinylation in this manner does not adversely and the complexes dissociate before the receptor can be affect GPCR function, allowing biotinylation to be used as purified from the extract. In this case, various chemical a chemical tag for surface receptors. Using variations of this basic biochemistry, it is possi- teraction with heterotrimeric G proteins (52) and with - ble to measure a wide variety of membrane trafficking pro- arrestins (53), and to examine the regulation of these protein cesses. For example, internalization of GPCRs has been interactions by ligand-induced activation of the receptor. Recent studies provide strong support for this idea and, specifically, provide evidence for homo- and heterodimeri- Methods for Examining Specific Protein zation of individual GPCRs in vivo. This principle is per- Interactions Involved in GPCR Function haps best established for receptor tyrosine kinases, where it and Regulation is well established that oligomerization of receptors is re- A salient lesson emerging from recent cell biological studies quired for appropriate ligand-dependent signal transduction is that GPCR signal transduction can be viewed, in essence, (54). A relatively early hint that GPCRs may also undergo as a dynamically regulated network of protein–protein in- oligomerization came from studies of the 2-adrenergic re- teractions that occur in specific subcellular locations. There- ceptor using epitope-tagging techniques, where it was ob- fore, an important goal of current and future research is to served that receptors tagged with one epitope could specifi- define these critical protein interactions and elucidate their cally coimmunoprecipitate receptors tagged with a distinct temporal and spatial regulation in intact cells and tissues. More recently, evidence for oligomerization of many Coimmunoprecipitation Techniques to GPCRs has been reported. A particularly compelling exam- Examine Defined Protein Interactions ple of this is the recent observation that distinct subtypes with GPCRs in Intact Cells of GABA-B receptor hetero-oligomerize in cells, and that As discussed above, it is possible to rapidly purify GPCRs oligomerization is essential for the formation of recombi- from cell or tissue extracts using receptor-specific antibodies nant receptors possessing the functional properties charac- 22: G-Protein–Coupled Receptors 285 teristic of native GABA-B receptors observed in vivo interactions. A cDNA library prepared from a tissue of inter- (57,58). Both the bait and prey expressed opioid receptors (59). In a recently published study transcription of the reporter gene. However, if the fused (60), glutathione S-transferase (GST)-fusion proteins en- bait and prey polypeptides form a sufficiently stable pro- coding the C-terminal tail of the D5 receptor were shown tein–protein interaction, they bring their corresponding to interact with the GABA-A receptor present in rat hippo- DNA binding and transcriptional activation domains into campal extracts. Additionally, using an antibody recogniz- close proximity, thus reconstituting transcriptional activa- ing the dopamine D5 receptor, it was possible to coimmu- tion of the reporter gene. Transformed yeast cells containing noprecipitate the GABA-A receptor from cell extracts. In addition to known proteins that mediate and regulate Protein interactions suggested to occur by the yeast two- GPCR signaling (heterotrimeric G proteins, GRKs, ar- hybrid system can be examined using various in vitro bio- restins), which were originally identified by functional as- chemical techniques, such as affinity chromatography facili- says using biochemical purification, cDNA cloning meth- tated by GST-fusion proteins. In addition to serving as an ods have facilitated the identification of additional protein independent assay for previously defined candidate interact- interactions with GPCRs that were completely unantici- ing proteins, this method can be used to identify novel pated (61). These novel protein interactions, while their protein interactions with GPCRs de novo (63). In this functional relevance remains unclear in many cases, are of method a DNA encoding a polypeptide sequence of interest great interest and potential therapeutic importance as drug is fused to GST using standard cDNA cloning techniques targets. The GST Of the many techniques for identifying novel pro- portion of the fusion protein allows the efficient immobili- tein–protein interactions developed over the last 10 years, zation of the protein by binding to agarose beads covalently interaction cloning methods such as the yeast two-hybrid derivatized with glutathione. Proteins from a cell or tissue system (62) have been particularly useful for studies of extract that bind to the fusion protein then can be isolated GPCRs. In the yeast two-hybrid system, protein interac- as an immobilized protein complex by affinity chromatogra- tions are detected by their ability to reconstitute the activity phy. A transcrip- shown recently (64) that the third cytoplasmic loop of the tion factor such as GAL4 can be divided into two domains: dopamine D2 receptor binds specifically to spinophilin, a a DNA binding domain and a transcriptional activation large cytoskeleton-associated protein that also binds to pro- domain. For the transcription factor to be active, these two tein phosphatase-1. A polypeptide sequence for which one phy using GST-fusion proteins. However, even in the event that extensive colocalization is observed, immunocytochem- ical techniques of this sort do not provide direct evidence for a physical interaction between candidate proteins. Coimmunoprecipitation techniques, as discussed above, A provide a useful method for addressing this question. How- ever, demonstrating that a specific protein association can occur in vivo is only the first step in the process of assessing the potential physiologic relevance of a novel protein inter- action, as this method generally does not provide any infor- mation regarding the possible functional activity of a candi- date protein interaction. Addressing this question can be a challenging task that involves creative application of diverse techniques and functional assays.

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