Female and male brains differ. Differences begin early during development due to a combination of genetic and hormonal events and continue throughout the lifespan of an individual. Although researchers from a myriad of disciplines are beginning to appreciate the importance of considering sex differences in the design and interpretation of their studies, this is an area that is full of potential pitfalls. A female's reproductive status and ovarian cycle have to be taken into account when studying sex differences in health and disease susceptibility, in the pharmacological effects of drugs, and in the study of brain and behavior. To investigate sex differences in brain and behavior there is a logical series of questions that should be answered in a comprehensive investigation of any trait. First, it is important to determine that there is a sex difference in the trait in intact males and females, taking into consideration the reproductive cycle of the female. Then, one must consider whether the sex difference is attributable to the actions of gonadal steroids at the time of testing and/or is sexually differentiated permanently by the action of gonadal steroids during development. To answer these questions requires knowledge of how to assess and/or manipulate the hormonal condition of the subjects in the experiment appropriately. This article describes methods and procedures to assist scientists new to the field in designing and conducting experiments to investigate sex differences in research involving both laboratory animals and humans.

Tacrolimus is the mainstay immunosuppressant drug used after solid organ and hematopoietic stem cell transplantation. Individuals who express CYP3A5 (extensive and intermediate metabolizers) generally have decreased dose-adjusted trough concentrations of tacrolimus as compared to those who are CYP3A5 non-expressers (poor metabolizers), possibly delaying achievement of target blood concentrations. We summarize evidence from the published literature supporting this association and provide dosing recommendations for tacrolimus based on CYP3A5 genotype when known (updates at www.pharmgkb.org).

Enhanced drug efflux mediated by ABCB1 P-glycoprotein and related ATP-binding cassette transporters is one of several mechanisms of multidrug resistance thought to impair chemotherapeutic success in human cancers. In malignant melanoma, its potential contribution to chemoresistance is uncertain. Here, we show that ABCB5, which functions as a determinant of membrane potential and regulator of cell fusion in physiologic skin progenitor cells, is expressed in clinical malignant melanoma tumors and preferentially marks a subset of hyperpolarized, CD133+ stem cell phenotypeexpressing tumor cells in malignant melanoma cultures and clinical melanomas. We found that ABCB5 blockade significantly reversed resistance of G3361 melanoma cells to doxorubicin, an agent to which clinical melanomas have been found refractory, resulting in a 43% reduction in the LD 50 from 4 to 2.3 Mmol/L doxorubicin (P < 0.05). Our results identified ABCB5-mediated doxorubicin efflux transport as the underlying mechanism of resistance, because ABCB5 blockade significantly enhanced intracellular drug accumulation. Consistent with this novel ABCB5 function and mechanism in doxorubicin resistance, gene expression levels of the transporter across a panel of human cancer cell lines used by the National Cancer Institute for drug screening correlated significantly with tumor resistance to doxorubicin (r = 0.44; P = 0.016). Our results identify ABCB5 as a novel drug transporter and chemoresistance mediator in human malignant melanoma. Moreover, our findings show that ABCB5 is a novel molecular marker for a distinct subset of chemoresistant, stem cell phenotype-expressing tumor cells among melanoma bulk populations and indicate that these chemoresistant cells can be specifically targeted via ABCB5 to enhance cytotoxic efficacy. (Cancer Res 2005; 65(10): 4320-33)

As a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) plays a key role in pain perception and addiction. Genetic variants of OPRM1 have been implicated in predisposition to drug addiction, in particular the single nucleotide polymorphism A118G, leading to an N40D substitution, with an allele frequency of 10 -32%, and uncertain functions. We have measured allele-specific mRNA expression of OPRM1 in human autopsy brain tissues, using A118G as a marker. In 8 heterozygous samples measured, the A118 mRNA allele was 1.5-2.5-fold more abundant than the G118 allele. Transfection into Chinese hamster ovary cells of a cDNA representing only the coding region of OPRM1, carrying adenosine, guanosine, cytidine, and thymidine in position 118, resulted in 1.5-fold lower mRNA levels only for OPRM1-G118, and more than 10-fold lower OPRM1 protein levels, measured by Western blotting and receptor binding assay. After transfection and inhibition of transcription with actinomycin D, analysis of mRNA turnover failed to reveal differences in mRNA stability between A118 and G118 alleles, indicating a defect in transcription or mRNA maturation. These results indicate that OPRM1-G118 is a functional variant with deleterious effects on both mRNA and protein yield. Clarifying the functional relevance of polymorphisms associated with susceptibility to a complex disorder such as drug addiction provides a foundation for clinical association studies.Drug addiction is a complex disorder with a strong genetic component (1, 2). Serving as a primary target for opioid drugs and peptides, the mu opioid receptor (OPRM1) mediates the effects of morphine and heroin (3, 4). By impinging on dopaminergic pathways, OPRM1 also plays a role in addiction to other drugs of abuse, such as cocaine, nicotine, and alcohol (5, 6).Because of its central role in drug addiction, numerous studies have addressed potential contributions of polymorphisms in the gene encoding OPRM1 to addiction susceptibility (7,8). Among multiple single nucleotide polymorphisms (SNPs) 2 in OPRM1, C17T (A6V), and A118G (N40D) are well studied nonsynonymous SNPs located at the N terminus of the receptor. In particular, A118G, with an allele frequency of 10 -32% in different ethnic groups (9), has been associated with susceptibility to heroin, nicotine, and alcohol addiction (10 -12). However, other studies have failed to corroborate these associations (13, 14), possibly as a result of population admixture.The A118G polymorphism has also been linked to differences in pharmacological properties of OPRM1. In a Swedish population, the G118 variant was shown to correlate with poor response to naltrexone in the treatment of alcoholism (15). Moreover, OPRM1-antagonist naloxone elicited an increased cortisol response in individuals with a G118 allele (16,17). In transfected cells, OPRM1-Asp 40 was reported to have 3-fold higher affinity for ␤-endorphin than OPRM1-Asn 40 (18), suggesting a gain of function, but subsequent studies have failed to corroborate these results...

Subcortical dopamine D2 receptor (DRD2) signaling is implicated in cognitive processes and brain disorders, but the effect of DRD2 variants remains ambiguous. We measured allelic mRNA expression in postmortem human striatum and prefrontal cortex and then performed single nucleotide polymorphism (SNP) scans of the DRD2 locus. A previously uncharacterized promoter SNP (rs12364283) located in a conserved suppressor region was associated with enhanced DRD2 expression, whereas previously studied DRD2 variants failed to affect expression. Moreover, two frequent intronic SNPs (rs2283265 and rs1076560) decreased expression of DRD2 short splice variant (expressed mainly presynaptically) relative to DRD2 long (postsynaptic), a finding reproduced in vitro by using minigene constructs. Being in strong linkage disequilibrium with each other, both intronic SNPs (but not rs12364283) were also associated with greater activity of striatum and prefrontal cortex measured with fMRI during working memory and with reduced performance in working memory and attentional control tasks in healthy humans. Our results identify regulatory DRD2 polymorphisms that modify mRNA expression and splicing and working memory pathways.allelic expression imbalance ͉ splice variant ͉ promoter polymorphism ͉ brain imaging A lthough genetic factors contribute to central nervous system (CNS) disorders, only a few genes have been identified as unequivocal risk factors. Aberrant subcortical dopamine D2 receptor (DRD2) signaling is implicated in brain disorders such as drug addiction (1, 2), schizophrenia, and Parkinson's disease (3, 4). DRD2 variants Taq1A, promoter polymorphism Ϫ141C del/ins, and a synonymous SNP in exon 7 (C957T) have been associated with schizophrenia and drug abuse (5-8), but associations are not consistently replicated (9-11). Moreover, polymorphisms relevant in vivo remain unknown. Our goal was to identify functional DRD2 polymorphisms linked to CNS functions.DRD2 variants could have maximal impact in the basal ganglia endowed with prominent DRD2 signaling. A crossroad between cortex and dopamine projections from the brainstem, basal ganglia in the caudate and pallidum mediate cognitive processes (12)(13)(14) and contribute to focus of working memory (15). Dopamine-DRD2 signaling in these structures decreases GABA (16, 17) and glutamate inputs to striatal spiny neurons (18). DRD2 density affects working memory performance in mice (19), and striatal DRD2 receptor availability is linked to working memory and attention in humans (20). Another mechanism modulating DRD2 signaling involves alternative splicing of exon 6 to yield DRD2L (long) and DRD2S (short, considered an autoreceptor), expressed mainly postsynaptically and presynaptically, respectively (21, 22). Relative expression of DRD2S and L is critical to dopamine modulation of GABA and glutamate striatal transmission (23, 24).We searched for genetic variants modulating DRD2 neurotransmission in human brain. Because the DRD2 locus lacks frequent nonsynonymous SNPs that alter receptor...