Breaking barriers in the genomics and pharmacogenetics of drug addiction.

Drug addiction remains a substantial health issue with limited treatment options currently available. Despite considerable advances in the understanding of human genetic architecture, the genetic underpinning of complex disorders remains elusive. On the basis of our current understanding of neurobiology, numerous candidate genes have been implicated in the etiology and response to treatment for different addictions. Genome-wide association (GWA) studies have also identified novel targets. However, replication of these studies is often lacking, and this complicates interpretation. The situation is expected to improve as issues such as phenotypic characterization, the apparent "missing heritability," the identification of functional variants, and possible gene-environment (G × E) interactions are addressed. In addition, there is growing evidence that genetic information can be useful in refining the choice of addiction treatment. As genetic testing becomes more common in the practice of medicine, a variety of ethical and practical challenges, some of which are unique to drug addiction, will also need to be considered.

Regulation of transcription factors by heterotrimeric G proteins.

Lessons from viral hijacks of cells and cancer biology suggest that the activation of G protein-coupled receptors (GPCRs) often results in the modulation of various transcription factors and cofactors. Since drugs acting on GPCRs represent a significant portion of therapeutic agents currently in use, it is important to understand the actions of GPCRs on gene expression. GPCRs and their associated heterotrimeric G proteins are known to regulate gene transcription through complex signaling networks. The G protein-mediated signaling cascades have been extensively studied and accumulating evidence indicates that the four subfamilies of G proteins may utilize both common and unique pathways for transcriptional regulation. This review aims to provide a contemporary account of our understanding on the regulation of transcription factors by GPCRs, with a special emphasis on specific regulations of transcription factors such as STAT3 and NF-kappaB by individual G protein subfamilies. Functional impacts of the signal integration between different pathways and the contributions by other GPCR-interacting molecules will also be briefly discussed.

Evaluation of replacing the existing diagnostic strategy for Neisseria gonorrhoeae and Chlamydia trachomatis infections with sole molecular testing of urine specimens in a sexually transmitted infection clinic setting.

OBJECTIVES: To evaluate nucleic acid testing of urine specimens against conventional Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) tests in genital swab specimens in a sexually transmitted infection (STI) clinic setting. METHODS: Genital swab and urine samples were collected from attendees of public STI clinics in Hong Kong from May to June 2007. Swab specimens were subjected to on-site Gram stained microscopy and inoculation onto modified Thayer-Martin medium for NG culture before laboratory processing. CT PCR on genital swabs was performed by the Roche Cobas Amplicor test. Urine samples were tested for CT and NG by the Aptima Combo 2 (AC2) assay. RESULTS: Data from 414 patients were analysed. The sensitivity and specificity of AC2 for NG were 100% (35/35) and 98.4% (373/379), respectively, with culture of genital swab specimens as standard. On-site microscopy provided timely results for empirical antimicrobial therapy, whereas culture yielded bacterial isolates for susceptibility testing and typing studies. Regarding CT, using Amplicor on genital swab specimens as reference, the sensitivity and specificity of AC2 were 98.7% (78/79) and 97.5% (313/321), respectively. Amplicor yielded uninterpretable results in 14 specimens due to PCR inhibitors. CONCLUSIONS: The current STI clinic and laboratory practices were practical and useful for clinical management, even though favourable results were obtained with the AC2 assay, which had streamlined laboratory workflow. The use of a molecular testing strategy may be cost-effective with microscopy and culture being targetted for patient groups with the highest expected yield of positive results.

Association of nicotine metabolite ratio and CYP2A6 genotype with smoking cessation treatment in African-American light smokers.

Cytochrome P450 2A6 (CYP2A6) is the main nicotine (NIC)-metabolizing enzyme in humans. We investigated the relationships between CYP2A6 genotype, baseline plasma trans- 3'-hydroxycotinine/cotinine (3HC/COT) (a phenotypic marker of CYP2A6 activity), and smoking behavior in African-American light smokers. Cigarette consumption, age of initiation, and dependence scores did not differ among 3HC/COT quartiles or CYP2A6 genotype groups. Slow metabolizers (SMs; both genetic and phenotypic) had significantly higher plasma NIC levels, suggesting that cigarette consumption was not reduced to adjust for slower rates of NIC metabolism. Individuals in the slowest 3HC/COT quartile had higher quitting rates with both placebo and NIC gum treatments (odds ratio 1.85, 95% confidence interval (CI) 1.08-3.16, P = 0.03). Similarly, the slowest CYP2A6 genotype group had higher quitting rates, although this trend did not reach significance (odds ratio 1.61, 95% CI 0.95-2.72, P = 0.08). The determination of the 3HC/COT ratio, and possibly CYP2A6 genotype, may be useful in the future for personalizing the choice of smoking cessation treatment in African-American light smokers.

Overview of the pharmacogenomics of cigarette smoking.

Cigarette smoking increases the risk of numerous health problems, including cancer, cardiovascular and pulmonary disorders, making smoking the leading cause of preventable death in the world. Nicotine is primarily responsible for the highly addictive properties of cigarettes. Although the majority of smokers express a desire to quit, few are successful in doing so. Twin and family studies have indicated substantial genetic contributions to smoking behaviors. One major research focus has been to elucidate the specific genes involved; this has been accomplished primarily through genome-wide linkage analyses and candidate gene association studies. Much attention has focused on genes involved in the neurotransmitter pathways for the brain reward system and genes altering nicotine metabolism. This paper reviews the current state of knowledge for genetic factors implicated in smoking behaviors, and examines how genetic variations may affect therapeutic outcomes for drugs used to assist smoking cessation.

Cerebral metabolism in major depression and obsessive-compulsive disorder occurring separately and concurrently.

BACKGROUND: The frequent comorbidity of major depressive disorder (MDD) and obsessive-compulsive disorder (OCD) suggests a fundamental relationship between them. We sought to determine whether MDD and OCD have unique cerebral metabolic patterns that remain the same when they coexist as when they occur independently. METHODS: [18F]-fluorodeoxyglucose positron emission tomography (PET) brain scans were obtained on 27 subjects with OCD alone, 27 with MDD alone, 17 with concurrent OCD+MDD, and 17 normal control subjects, all in the untreated state. Regional cerebral glucose metabolism was compared between groups. RESULTS: Left hippocampal metabolism was significantly lower in subjects with MDD alone and in subjects with concurrent OCD+MDD than in control subjects or subjects with OCD alone. Hippocampal metabolism was negatively correlated with depression severity across all subjects. Thalamic metabolism was significantly elevated in OCD alone and in MDD alone. Subjects with concurrent OCD+MDD had significantly lower metabolism in thalamus, caudate, and hippocampus than subjects with OCD alone. CONCLUSIONS: Left hippocampal dysfunction was associated with major depressive episodes, regardless of primary diagnosis. Other cerebral metabolic abnormalities found in OCD and MDD occurring separately were not seen when the disorders coexisted. Depressive episodes occurring in OCD patients may be mediated by different basal ganglia-thalamic abnormalities than in primary MDD patients.

Regional brain metabolic changes in patients with major depression treated with either paroxetine or interpersonal therapy: preliminary findings.

BACKGROUND: In functional brain imaging studies of major depressive disorder (MDD), regional abnormalities have been most commonly found in prefrontal cortex, anterior cingulate gyrus, and temporal lobe. We examined baseline regional metabolic abnormalities and metabolic changes from pretreatment to posttreatment in subjects with MDD. We also performed a preliminary comparison of regional changes with 2 distinct forms of treatment (paroxetine and interpersonal psychotherapy). METHODS: Twenty-four subjects with unipolar MDD and 16 normal control subjects underwent resting F 18 ((18)F) fluorodeoxyglucose positron emission tomography scanning before and after 12 weeks. Between scans, subjects with MDD were treated with either paroxetine or interpersonal psychotherapy (based on patient preference), while controls underwent no treatment. RESULTS: At baseline, subjects with MDD had higher normalized metabolism than controls in the prefrontal cortex (and caudate and thalamus), and lower metabolism in the temporal lobe. With treatment, subjects with MDD had metabolic changes in the direction of normalization in these regions. After treatment, paroxetine-treated subjects had a greater mean decrease in Hamilton Depression Rating Scale score (61.4%) than did subjects treated with interpersonal psychotherapy (38.0%), but both subgroups showed decreases in normalized prefrontal cortex (paroxetine-treated bilaterally and interpersonal psychotherapy-treated on the right) and left anterior cingulate gyrus metabolism, and increases in normalized left temporal lobe metabolism. CONCLUSIONS: Subjects with MDD had regional brain metabolic abnormalities at baseline that tended to normalize with treatment. Regional metabolic changes appeared similar with the 2 forms of treatment. These results should be interpreted with caution because of study limitations (small sample size, lack of random assignment to treatment groups, and differential treatment response between treatment subgroups).

G(z) signaling: emerging divergence from G(i) signaling.

A large variety of neurotransmitters, hormones, and chemokines regulate cellular functions via cell surface receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins) belonging to the G(i) subfamily. All members of the G(i) subfamily, with the sole exception of G(z), are substrates for the pertussis toxin ADP-ribosyl transferase. G(z) also exhibits unique biochemical and regulatory properties. Initial portrayals of the cellular functions of G(z) bear high resemblance to those of other G(i) proteins both in terms of the receptors and effectors linked to G(z). However, recent discoveries have begun to insinuate a distinct role for G(z) in cellular communication. Functional interactions of the alpha subunit of G(z) (Galpha(z)) with the NKR-P1 receptor, Galpha(z)-specific regulator of G protein signaling, p21-activated kinase, G protein-regulated inducers of neurite outgrowth, and the Eya2 transcription cofactor have been demonstrated. These findings provide possible links for G(z) to participate in cellular development, survival, proliferation, differentiation and even apoptosis. In this review, we have drawn a sketch of a signaling network with G(z) as the centerpiece. The emerging picture is one that distinguishes G(z) from other members of the G(i) subfamily.

Galpha(14) links a variety of G(i)- and G(s)-coupled receptors to the stimulation of phospholipase C.

1. The bovine Galpha(14) is a member of the G(q) subfamily of G proteins that can regulate phospholipase Cbeta isoforms but the extent to which Galpha(14) recognizes different receptor classes is not known. 2. Galpha(14) was cotransfected with a variety of receptors in COS-7 cells, and agonist-induced stimulation of phospholipase C was then measured. 3. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Galpha(14) stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. 4. Among the three opioid receptors (mu, delta, and kappa), only the delta receptor was capable of stimulating IP formation when coexpressed with Galpha(14) in COS-7 cells. 5. A panel of G(i)- and G(s)-linked receptors was screened for their ability to stimulate IP accumulation via Galpha(14). The adenosine A(1), complement C5a, dopamine D(1), D(2) and D(5), formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Galpha(14), while the alpha(2)- and beta(2)-adrenoceptors were able to do so. 6. Galpha(14)-mediated stimulation of phospholipase Cbeta was agonist dose-dependent. These data demonstrate that although Galpha(14) can interact with different classes of receptors, it is much less promiscuous than Galpha(15) or Galpha(16).

The amino terminus of Galpha(z) is required for receptor recognition, whereas its alpha4/beta6 loop is essential for inhibition of adenylyl cyclase.

G(z) couples to most of the known G(i)-linked receptors and its alpha subunit (Galpha(z)) inhibits adenylyl cyclases as efficiently as Galpha(i) subtypes. A series of chimeric Galpha subunits with different portions of Galpha(z) and Galpha(t1) (a regulator of cGMP phosphodiesterase) were constructed to study the essential structural elements of Galpha(z) that determine receptor coupling and effector interaction. The receptor-mediated functions of the chimeras were assessed in two aspects: 1) stimulation of type 2 adenylyl cyclase through the release of betagamma subunits from the chimeras, and 2) inhibition of isoproterenol-stimulated adenylyl cyclase by the chimeric Galpha subunits. The results suggested that the presence of both termini of Galpha(z) were critical for coupling to delta-opioid receptor, with the N-terminal region being more important. Moreover, a stretch of amino acids (295-319) corresponding to the alpha4/beta6 loop was identified as one of the adenylyl cyclase inhibitory domains of Galpha(z).

Preactivation permits subsequent stimulation of phospholipase C by G(i)-coupled receptors.

In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where G(i)-linked receptors augment G(q)-dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase Cbeta (PLCbeta) via the G protein betagamma-dimers. Herein, we explored the possibility that augmentation by betagamma-dimers requires preactivation of PLCbeta. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The G(i)-coupled delta- and kappa-opioid receptors could not stimulate PLCbeta unless they were coexpressed with Galpha(16). The opioid-induced response was dose-dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of betagamma-subunits released from the G(i) proteins. When PLCbeta was preactivated by constitutively active mutants of Galpha(16), Galpha(q), or Galpha(14), opioids enhanced the activity by 80 to 300% and such responses were mostly pertussis toxin-sensitive. The opioid-induced enhancement was dose-dependent and could not be blocked by staurosporin, a protein kinase C inhibitor. Other G(i)-coupled receptors that were ineffective on their own also acquired the ability to stimulate PLCbeta in the presence of a constitutively active mutant of Galpha(q). Coactivation of endogenous or exogenous G(q)-coupled receptors with the delta-opioid receptor produced strong stimulations of PLCbeta and such responses could be partially blocked by pertussis toxin. These results show that enhancement of G(q)-dependent signals by G(i)-coupled receptors requires activated PLCbeta and is mediated via the betagamma-dimer.

The effect of protein kinase C activation on G(z)-mediated regulation of type 2 and 6 adenylyl cyclases.

Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2.

Incorporation of Galpha(z)-specific sequence at the carboxyl terminus increases the promiscuity of galpha(16) toward G(i)-coupled receptors.

Although the promiscuous nature of G(16) allows it to interact with numerous G protein-coupled receptors, several G(i)-linked receptors are incapable of activating phospholipase C via G(16). A series of chimeras between Galpha(16) and Galpha(z) were constructed and assayed for their ability to mediate receptor-induced stimulation of phospholipase C. Two Galpha(16/z) chimeras harboring 25 or 44 Galpha(z)-specific sequences at their C termini (named 16z25 and 16z44) were capable of responding to 14 different G(i)-coupled receptors tested, including those that were either unable to associate with Galpha(16) (melatonin Mel1c) or activate Galpha(16) weakly (micro-opioid and type 1 somatostatin). Agonist-induced stimulation of phospholipase C was more efficiently mediated (higher maximal and lower EC(50) value) by 16z44 than by Galpha(16). Both 16z25 and 16z44 were also coupled to G(s)- and G(q)-linked receptors. Incorporation of Galpha(z) sequence at the N terminus of Galpha(16) did not further enhance the ability of the chimeras to interact with G(i)-coupled receptors. Expression of the various chimeras was verified by immunodetection and functional analysis of their constitutively activated mutants. These results show that the incorporation of alpha4/beta6 and alpha5 regions of Galpha(z) into a Galpha(16) backbone can improve the recognition of G(i)-coupled receptors. Galpha(16/z) chimeras with expanded capability to interact with G(i)-linked receptors may be used to link orphan receptors to the stimulation of phospholipase C.

Disruption of receptor-mediated activation of G protein by mutating a conserved arginine residue in the switch II region of the alpha subunit.

A naturally occurring point mutation (R231H) within one of the major 3gamma-binding surface (switch II region) on the a subunit of Gs (alpha(s)) has previously been found to disrupt receptor-mediated activation of Gs. The disruption caused by mutating this conserved residue may be a general phenomenon for all a subunits. Homologous mutants of the alpha subunit of Gz [alpha(z); a negative regulator of adenylyl cyclase (AC)] and G16 (alpha16; a stimulator of phospholipase C) were constructed and examined for receptor-mediated regulation of their corresponding effectors. The mutant alphazR209H cannot be fully activated by the delta-opioid receptor, as indicated by the impairment of the inhibition of alpha(s)-stimulated AC and betagamma-mediated stimulation of AC type II (AC2). Similarly, the mutant alpha16R216H lost the ability to mediate receptor-induced activation of phospholipase C and AC2. The receptor coupling efficacy and promiscuity of alpha16R216H were eradicated. The mutation of the conserved arginine has no observable effect on the constitutive activities of the GTPase-deficient derivatives of both alpha(z) and alpha16. The alpha subunit of Gt1 (transducin; alphat1) attenuated betagamma-mediated stimulation of AC2 by sequestrating free betagamma subunits, but the mutant alphat1R204H showed reduced ability to scavenge betagamma-mediated AC2 activation. Presumably, mutation of the conserved arginine disrupted the subunit interactions in addition to the impairment of receptor interaction.

Alanine-23 of transducin alpha subunit is involved in defining the affinity for betagamma complex.

Transducin a subunit (alpha(t1)) shows extraordinarily high affinity to G protein beta(gamma) complex. One of the beta(gamma)-binding regions on alpha(t1) is the amino-terminal helix. Alanine-23 is uniquely found on alpha(t1) but not other members of the G(i)-subfamily. Mutation of alanine-23 into serine reduced the ability of alpha(t1) to sequester beta(gamma)-mediated stimulation of type II adenylyl cyclase. The functional impairment is independent to the protein expression levels. Molecular modeling indicated that the hydrophobic interaction between the side chains of alanine-23 of alpha(t1) and leucine-55 of the beta1 subunit could be disrupted by the introduction of a hydroxyl group. This study showed that alanine-23 of alpha(t1) is probably involved in defining its affinity for the beta(gamma) complex.