By Q. Hamlar. Arizona State University.

However discount 60 ml rogaine 5 fast delivery, mutations that constitutively activate receptors and the deletion of limited regions in extracellular or cytoplasmic identification of functional mutant receptors predicted to domains are often well tolerated and have been quite in- contain fewer than seven transmembrane domains (31) generic rogaine 5 60 ml on line. For example, deletion of residues located in the amino-terminal extracellular domain of polypeptide recep- Use of Biophysical Approaches tors [such as the follicle-stimulating hormone (FSH) recep- tor] and the calcium receptor implicate this domain in li- Biophysical techniques are essential for detailed examina- gand interaction. Deletion of residues located in the third tion of protein structure and conformational change. One cytoplasmic loop of various receptors, such as the musca- reason these methodologies have had limited application in rinic acetylcholine receptors, implicated this domain in the study of GPCRs is that they typically require milligram functional coupling to heterotrimeric G proteins (27). For many years rhodopsin, purified from retina, was the only GPCR that could be generated Substitution or Chimeric Mutagenesis in sufficient quantity for biophysical study. Indeed, much of A very powerful approach to site-directed mutagenesis is to what we know about GPCR structure and conformational substitute entire series of residues from one receptor with change has been elucidated from elegant biophysical studies the corresponding residues of another. Recently, the development of improved based on the idea that receptors are composed of modular expression and purification strategies have made it possible structural domains, and takes advantage of the fact that to obtain other GPCRs in sufficient quantity and purity receptor domains that mediate similar functions often have for biophysical study. Thus it is likely that biophysical ap- conserved amino acid sequence. Chimeric substitutions are proaches will play an increasingly important role in future often less disruptive than deletions to the overall structure studies of GPCR structure and activation. For example, chimeric mutagenesis has been useful for defining transmembrane residues that Structural Studies of Rhodopsin mediate subtype-specific and species-specific differences in ligand binding to adrenergic receptors. Receptor chimeras High-resolution structural information can be provided by between 2- and 2-adrenergic receptors defined multiple x-ray diffraction methodologies applied to ordered three- cytoplasmic domains that contribute to the specificity of dimensional crystals of pure protein. Rhodopsin, a GPCR receptor interaction with their cognate heterotrimeric G mediating phototransduction in the retina, has been a favor- proteins (4). Previous stud- ies using electron diffraction of two-dimensional crystals of Use of Random Mutagenesis rhodopsin obtained structural information to a resolution of In contrast to site-directed mutagenesis, random mutagene- approximately 7. Recently x-ray diffraction has 22: G-Protein–Coupled Receptors 281 been used to solve the structure of three dimensional crystals chemical probe. Approaches of this type have been applied of rhodopsin to a resolution of 2. This accomplishment˚ to several GPCRs, and have begun to yield interesting new is truly a major milestone in the field, revealing for the information about the dynamic effects of clinically relevant first time the atomic structure of any GPCR and providing drugs on GPCR structure (29). A precedent for such an approach is the membrane structure are relevant to other GPCRs. It remains to be determined cation of existing drugs. However, an important goal is to the degree to which specific features of diverse GPCRs are design completely new drugs de novo based on the struc- actually conserved at the level of atomic resolution. A clue that this may be based on well established differences in the pharmacology of possible comes from recent studies of mutant GPCRs, in individual GPCRs, one might expect there to be significant which histidine residues have been introduced at defined limitations of such homology-based predictive methods, at positions in the receptor structure that can be coordinated least with respect to structural features involved in drug by certain metal ions. Nevertheless, the available experimental data leave receptor, by coordinating histidine residues introduced little doubt that this approach is an important starting point within specific transmembrane helices, influences the recep- for mechanistic studies and for rational drug development tor conformation to either activate or inactivate the receptor (34). While it is unlikely that this strategy will directly yield clinically Biophysical Studies of Conformational useful drugs, these exciting studies serve as a proof of the Dynamics Involved in GPCR Activation principle motivating further studies of GPCR structure and While crystallographic methods have the potential to pro- conformational change. Thus REGULATION OF RECEPTOR SIGNALING additional methods are required to examine dynamic con- Methods to Examine Regulation of formational transitions that mediate ligand-dependent sig- Receptors byPosttranslational nal transduction via GPCRs. Several biophysical approaches Modification: GPCR Phosphorylation have been utilized for this purpose. Classic studies of rho- dopsin measured the optical absorbance properties of this Many different types of posttranslational modification have photoprotein that are highly sensitive to changes in protein been implicated in the regulation in of GPCR function, conformation. Sophisticated studies using optical spectros- localization or stability. A detailed discussion of this large copy indicate that rhodopsin cycles rapidly through a series area of research is beyond the scope of this chapter. Instead, of distinct conformational states following photon-induced we illustrate the use of specific methods by discussing some activation. Many other types of biophysical techniques have aspects of protein phosphorylation, the most extensively been applied to examine specific features of light-induced characterized type of posttranslational modification that conformational changes of rhodopsin, as well as to examine regulates GPCRs. Specific residues in the receptor protein can be labeled 1950s demonstrated that enzyme-catalyzed protein phos- with a chemical probe, typically using a combination of phorylation and dephosphorylation reactions were involved site-directed mutagenesis and organic chemistry techniques. Phosphorylation of mammalian proteins in relative size and charge. Proteins resolved by gel electrophoresis can be transferred Serine/threonine phosphorylation is widely recognized to to a membrane composed of nitrocellulose or polyvinyl di- regulate GPCRs. Tyrosine phosphorylation, a more recently fluoride (PVDF). This allows many manipulations to be discovered modification that is well established to mediate performed, such as detection of a specific protein from a signaling via non-GPCR growth factor receptors (39), may complex mixture by the ability of the protein to be bound also play a role in regulating certain GPCRs (40).

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GFR— glom erular filtration rate; RBF— renal blood flow; m edium –induced renal dysfunction order 60 ml rogaine 5 visa. Based on experim ental m od- TH — Tam m H orsfall protein buy discount rogaine 5 60 ml on line. Thus it is im portant to select the least invasive diagnostic proce- dure that provides the m ost inform ation, so that the patient can PREVENTION OF CONTRAST m ake an inform ed choice from the available clinical alternatives. ASSOCIATED NEPHROPATHY Since radiographic contrast im aging is frequently perform ed for diabetic nephropathy, congestive heart failure, or chronic renal failure, concurrent adm inistration of renoprotective Hydrate patient before the study (1. The correction of Use nonionic, iso-osmolar contrast media for patients at high risk (see Figure 11-21). Lim iting the total volum e of contrast m edium and using nonionic, isoosm olar FIGURE 11-23 m edia have proven to be protective for high-risk patients. The goal of m an- Pretreatm ent with calcium antagonists is an intriguing but agem ent is the prevention of contrast-associated nephropathy. Bennett W M , Porter GA: O verview of clinical nephrotoxicity. Thadhani R, Pascual M , Bonventre JV: Acute renal failure. Vestergaard P, Am disen A, H ansen AE, Schou M : Lithium treatm ent M ed 1996, 334:1448–1460. De Broe M E: Prevention of am inoglycoside nephrotoxicity. London:BailliËre treatm ent: initial and follow-up tests in m anic-depressive patients. Lietm an PS: Am inoglycosides and spectinoycin: am inocylitols. Edited by and non–lithium -treated patients with affective disorders. N ew York: John W iley & Psychiatry Scand 1980; 62:343–355. Battle DC, Dorhout-M ees EJ: Lithium and the kidney. Kaloyanides GJ, Pastoriza-M unoz E: Am inoglycoside nephrotoxicity. Cell biology of am inoglycoside nephrotoxicity: newer Kluwer Academ ic, 1998:383–395. J Pharm acol Exp or renal artery stenosis in a solitary kidney. Giuliano RA, Verpooten GA, De Broe M E: The effect of dosing strat- 27. Textor SC: ACE inhibitors in renovascular hypertension. Cardiovasc egy on kidney cortical accum ulation of am inoglycosides in rats. In Clinical nephrotoxins— renal injury from drugs schedule decreases the accum ulation of gentam icin and netilm icin in and chem icals. Edited by De Broe M E, Porter GA, Bennett W M , the renal cortex of hum ans. De Broe M E, Verbist L, Verpooten GA: Influence of dosage schedule 29. Sm ith W R, N eil J, Cusham W C, Butkus DE: Captopril associated on renal cortical accum ulation of am ikacin and tobram ycin in m an. O pie LH : Angiotensin-converting enzym e inhibitors. W helton A, W atson J: N onsteroidal anti-inflam m atory drugs: effects peak serum levels from renal failure. Edited by De Broe M E, Porter GA, Bennett ty in patients treated with am inoglycosides. Zager RA: A focus of tissue necrosis increases renal susceptibility to 1998, 338:446–452. M itchell JA, Akarasereenont P, Thiem erm ann C, et al. Andreoli TE: O n the anatom y of am photericin B-cholesterol pores in nonsteroidal antiinflam m atory drugs as inhibitors of constitutive and lipid bilayer m em branes. N ephrotoxins— Renal Injury From D rugs and Chem icals. Bennett W M , H enrich W L, Stoff JS: The renal effects of nonsteroidal De Broe M E, Porter GA, Bennett W M , Verpooten GA. Dordrecht: anti-inflam m atory drugs: sum m ary and recom m endations. Bennett W M : M echanism s of acute and chronic nephrotoxicity from 35. H eym an SN , Rosen S, Brezis M : Radiocontrast nephropathy: a para- im m unosuppressive drugs.

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In consensual the assault discount 60 ml rogaine 5 amex, survivor order rogaine 5 60 ml overnight delivery, or assailant that might increase risk for sex, the risk for HIV transmission from vaginal intercourse HIV transmission. Te risk for HIV transmission from oral sex is substan- discussed with the patient: 1) the unproven benefit and tially lower. Site of exposure to ejaculate, viral load in ejaculate, and potential benefts (i. Providers should emphasize that PEP survivor also might increase the risk for HIV. Clinical man- the sexual abuse of children is frequently associated with mul- agement of the survivor should be implemented according to tiple episodes of assault and might result in mucosal trauma the following guidelines (78). Specialist consultation on PEP (see Sexual Assault or Abuse of Children). Te sooner PEP reduced risk for acquiring HIV in a study of health-care work- is initiated after the exposure, the higher the likelihood that ers who had percutaneous exposures to HIV-infected blood it will prevent HIV transmission if HIV exposure occurred; (480). On the basis of these results and the results of animal however, distress after an assault also might prevent the survivor studies, PEP has been recommended for health-care workers from accurately weighing exposure risks and benefts of PEP who have occupational exposures to HIV (446). Tese fnd- and from making an informed decision to start such therapy. If HIV exposure has occurred, be ofered a 3–5-day supply of PEP, and a follow-up visit initiation of PEP as soon as possible after the exposure likely should be scheduled several days later to allow for additional increases beneft. Although a defnitive statement of beneft counseling. Te possible beneft §§ Hours of Sexual Assault of PEP in preventing HIV infection also should be discussed • Assess risk for HIV infection in the assailant. Implications of commonly encountered sexually trans- mitted (ST) or sexually associated (SA) infections for diagnosis and provide enough medication to last until the next return reporting of sexual abuse among infants and pre-pubertal children visit; reevaluate the survivor 3–7 days after initial assess- Evidence for ment and assess tolerance of medications. ST/SA confrmed sexual abuse Suggested action • If PEP is started, perform CBC and serum chemistry at † Gonorrhea* Diagnostic Report baseline (initiation of PEP should not be delayed, pend- Syphilis* Diagnostic Report† Human immunodefciency virus§ Diagnostic Report† ing results). Chlamydia trachomatis* Diagnostic Report† • Perform HIV antibody test at original assessment; repeat Trichomonas vaginalis Highly suspicious Report† at 6 weeks, 3 months, and 6 months. Condylomata acuminata Suspicious Report† (anogenital warts)* Genital herpes* Suspicious Report†¶ Sexual Assault or Abuse of Children Bacterial vaginosis Inconclusive Medical follow-up Recommendations in this report are limited to the identif- Source: Adapted from Kellogg N, American Academy of Pediatrics Committee on Child Abuse and Neglect. Te ¶ Unless there is a clear history of autoinoculation. Postnatally acquired gonorrhea; syphilis; ered if no conclusive explanation for nonsexual transmission and nontransfusion, nonperinatally acquired HIV are usually of an STD can be identifed. Sexual abuse should be suspected when genital herpes is diagnosed. Te investigation of sexual Reporting abuse among children who have an infection that could have All U. Although the exact requirements with recommendations by clinicians who have experience and difer by state, if a health-care provider has reasonable cause training in all elements of the evaluation of child abuse, neglect, to suspect child abuse, a report must be made. Te social signifcance of an infection that might providers should contact their state or local child-protection have been acquired sexually and the recommended action service agency regarding child-abuse reporting requirements regarding reporting of suspected child sexual abuse varies by in their states. In all Diseases cases in which an STD has been diagnosed in a child, eforts should be made to detect evidence of sexual abuse, including Examinations of children for sexual assault or abuse should conducting diagnostic testing for other commonly occurring be conducted in a manner designed to minimize pain and STDs (484–486). Collection of vaginal specimens in pre- Te general rule that sexually transmissible infections pubertal children can be very uncomfortable and should be beyond the neonatal period are evidence of sexual abuse has performed by an experienced clinician to avoid psychological exceptions. Te decision to obtain genital trachomatis among young children might be the result of or other specimens from a child to conduct an STD evaluation perinatally acquired infection and has, in some cases, persisted must be made on an individual basis. Genital warts have been diagnosed in place children at high-risk for STDs and constitute a strong children who have been sexually abused, but also in children indication for testing. BV • Te child has or has had symptoms or signs of an STD has been diagnosed in children who have been abused, but its or of an infection that can be sexually transmitted, even presence alone does not prove sexual abuse. In addition, most in the absence of suspicion of sexual abuse. Among the HBV infections in children result from household exposure signs that are associated with a confrmed STD diagnosis to persons who have chronic HBV infection. Cervical specimens are not rec- for other common STDs before the initiation of any treat- ommended for prepubertal girls. For boys with a urethral ment that could interfere with the diagnosis of those other discharge, a meatal specimen discharge is an adequate STDs. Because of the legal and psychosocial consequences substitute for an intraurethral swab specimen. Because of a false-positive diagnosis, only tests with high specifcities of the legal implications of a diagnosis of N. Te potential beneft to the child of a reliable infection in a child, if culture for the isolation of N. Gram stains are inadequate to ers with experience in the evaluation of sexually abused and evaluate prepubertal children for gonorrhea and should assaulted children. Specimens Te scheduling of an examination should depend on the from the vagina, urethra, pharynx, or rectum should be history of assault or abuse. If the initial exposure was recent, streaked onto selective media for isolation of N.

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Haplotype relative mine D4 receptors are supersensitive to ethanol rogaine 5 60 ml low cost, cocaine generic rogaine 5 60 ml with amex, and risk study of catechol-O-methyltransferase (COMT) and atten- methamphetamine. Dopamine D4 recep- high-enzyme activity Val allele with ADHD impulsive-hyperac- tor-knock-out mice exhibit reduced exploration of novel stimuli. Association of atten- chol-O-methyltransferase (COMT) gene polymorphism and at- tion deficit disorder and the dopamine transporter gene. Am J tention deficit hyperactivity disorder (ADHD) in an Irish sam- Med Genet 1995;56:993–998. No association between low between attention deficit hyperactivity disorder and a dopamine and high activity catecholamine-methyl-transferase (COMT) transporter polymorphism. Linkage study of catechol- hyperactivity disorder in children: heterogeneity owing to diag- O-methyltransferase and attention-deficit hyperactivity disor- nostic subtype and severity. A molecular genetic study deficit disorder and the DXS7 locus. Am J Med Genet 2000; of hyperkinetic disorder/attention deficit hyperactivity disorder. Association of the dopamine trans- effect of three noradenergic genes (ADRA2A, ADRA2C, DBH) porter gene (DAT1) with poor methylphenidate response [see on attention-defecit hyperactivity disorder and learning disabili- Comments]. J Am Acad Child Adolesc Psychiatry 1999;38: ties an Tourette syndrome subjects. No association of a ference to cocaine and amphetamine in mice lacking the dopa- tyrosine hydroxylase gene tetranucleotide repeat polymorphism mine transporter. Coloboma mouse mutant as an animal model of homeostasis. Differential regulation rosci Biobehav Rev 2000;24:51–57. The effect of sugar on behav- porter is required for in vivo MPTP neurotoxicity: evidence ior or cognition in children. The neuropsychiatric implications of low level 1322–1325. Controlled trial of methylphenidate in preschool D2 receptor locus as a modifying gene in neuropsychiatric disor- children with minimal brain dysfunction. Pregnancy delivery impairment in mice lacking dopamine D2 receptors. Nature and infancy complications and ADHD: issues of gene-environ- 1995;377:424–428. Sprich-Buckminster S, Biederman J, Milberger S, et al. Are in D2 dopamine receptor-deficient mice is determined by gene perinatal complications relevant to the manifestation of ADD? Evaluating the signifi- tor-deficient mice exhibit decreased dopamine transporter func- cance of minimal brain dysfunction: results of an epidemiologic tion but no changes in dopamine release in dorsal striatum. Abnormal synaptic plastic- and normal children: prenatal, developmental, and health his- ity in the striatum of mice lacking dopamine D2 receptors. Marital discord and child behavior prob- tors and the risk of subsequent referral for hyperactivity. J Abnorm Child Psychol 1990;18: Psychol Psychiatry 1992;33:1077–1090. Mother-child interactions, family conflicts and maternal Teratology 1979;19:119. Hyperkinesis and maternal butes that predict resilient outcomes. The effects of maternal depression on during a defined period in neonatal life induces permanent children. Depressed mothers as informants about their chil- mice. Effect of maternal nicotine on the develop- 1992;112:485–499. Downregulation of nicotinic chiatry Res 1986;17:241–246. Focal cerebral hypoperfusion Exp Ther 1993;266:1268–1276. Impact of fetal nicotine expo- Arch Neurol 1984;41:825–829. J Learn Disabil 1991;24: infantile autism and other types of childhood psychoses. Season of birth and neurode- in children with attention-deficit hyperactivity disorder. J Child velopmental disorders: summer birth is associated with dyslexia.

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