A polymorphism in the OPRM1 3'-untranslated region is associated with methadone efficacy in treating opioid dependence.

The µ-opioid receptor (MOR) is the primary target of methadone and buprenorphine. The primary neuronal transcript of the OPRM1 gene, MOR-1, contains a ~13 kb 3' untranslated region with five common haplotypes in European-Americans. We analyzed the effects of these haplotypes on the percentage of opioid positive urine tests in European-Americans (n=582) during a 24-week, randomized, open-label trial of methadone or buprenorphine/naloxone (Suboxone) for the treatment of opioid dependence. A single haplotype, tagged by rs10485058, was significantly associated with patient urinalysis data in the methadone treatment group. Methadone patients with the A/A genotype at rs10485058 were less likely to have opioid-positive urine drug screens than those in the combined A/G and G/G genotypes group (relative risk=0.76, 95% confidence intervals=0.73-0.80, P=0.0064). Genotype at rs10485058 also predicted self-reported relapse rates in an independent population of Australian patients of European descent (n=1215) who were receiving opioid substitution therapy (P=0.003). In silico analysis predicted that miR-95-3p would interact with the G, but not the A allele of rs10485058. Luciferase assays indicated miR-95-3p decreased reporter activity of constructs containing the G, but not the A allele of rs10485058, suggesting a potential mechanism for the observed pharmacogenetic effect. These findings suggest that selection of a medication for opioid dependence based on rs10485058 genotype might improve outcomes in this ethnic group.

Functional genetic variants in the vesicular monoamine transporter 1 modulate emotion processing.

Emotional behavior is in part heritable and often disrupted in psychopathology. Identification of specific genetic variants that drive this heritability may provide important new insight into molecular and neurobiological mechanisms involved in emotionality. Our results demonstrate that the presynaptic vesicular monoamine transporter 1 (VMAT1) Thr136Ile (rs1390938) polymorphism is functional in vitro, with the Ile allele leading to increased monoamine transport into presynaptic vesicles. Moreover, we show that the Thr136Ile variant predicts differential responses in emotional brain circuits consistent with its effects in vitro. Lastly, deep sequencing of bipolar disorder (BPD) patients and controls identified several rare novel VMAT1 variants. The variant Phe84Ser was only present in individuals with BPD and leads to marked increase monoamine transport in vitro. Taken together, our data show that VMAT1 polymorphisms influence monoamine signaling, the functional response of emotional brain circuits and risk for psychopathology.

The CHRNA5-A3-B4 gene cluster in nicotine addiction.

Nicotine addiction (NA) is a common and devastating disease, such that the annual number of deaths (world-wide) from tobacco-related diseases will double from 5 million in the year 2000 to 10 million in 2020. Nicotine is the only substance in tobacco which animals and humans will self-administer. NA, as a lifetime diagnosis, has been assessed in various approaches, including the concept of cigarettes per day (CPD). Other assessments of NA are somewhat more comprehensive, such as the Fagerstrom Test for Nicotine Dependence or the American Psychiatric Association's Diagnostic and Statistical Manual (fourth edition) diagnosis of nicotine dependence. These different measures have moderate agreement with one another. Twin, family and adoption studies have shown that these different assessments of NA have substantial heritability (that fraction of risk attributable to genetic factors). The heritability of NA has been estimated at 50-75%, depending on the definition and the population under study. DNA-based studies of NA have been somewhat successful in identifying a common haplotype, which increases risk for NA among European-origin populations. This haplotype explains a small amount of variance, accounting for ∼1 CPD, and it includes the α5 and the α3 nicotinic receptor subunit genes (CHRNA5 and CHRNA3). The review will focus on this implicated region. In this risk region, there is a common (among European-origin people) mis-sense single-nucleotide polymorphism in the CHRNA5 gene (D398N), which changes a conserved amino acid from aspartic acid to asparagine. The risk allele (398N) confers decreased calcium permeability and more extensive desensitization, according to in vitro cellular studies, raising the possibility that a positive allosteric modulator of the (α4ß2)(2)α5 type of nicotinic receptor might have therapeutic potential in NA. There are other genetic influences on NA in this region, apart from the mis-sense variant, and additional biological experiments must be done to understand them.

Confirmation of multiple seizure susceptibility QTLs on chromosome 15 in C57BL/6J and DBA/2J inbred mice.

To confirm seizure susceptibility (SZS) quantitative trait loci (QTLs) on chromosome (chr) 15 identified previously using C57BL/6J (B6) and DBA/2J (D2) mice and to refine their genomic map position, we studied a set of three congenic strains in which overlapping segments of chr 15 from D2 were transferred onto the B6 background. We measured thresholds for generalized electroshock seizure (GEST) and maximal electroshock seizure (MEST) in congenic strains and B6-like littermates and also tested their responses to kainic acid (KA) and pentylenetetrazol (PTZ). Results document that MEST is significantly lower in strains 15M and 15D, which harbor medial and distal (telomeric) segments of chr 15 (respectively) from D2, compared with strain 15P, which harbors the proximal (acromeric) segment of chr 15 from D2, and with control littermates. Congenic strains 15P and 15M exhibited greater KA SZS compared with strain 15D and B6-like controls. All congenic strains were similar to controls with regard to PTZ SZS. Taken together, results suggest there are multiple SZS QTLs on chr 15 and that two QTLs harbor gene variants that affect MEST and KA SZS independently. The MEST QTL is refined to a 19 Mb region flanked by rs13482630 and D15Mit159. This interval contains 350 genes, 183 of which reside in areas where the polymorphism rate between B6 and D2 is high. The KA QTL interval spans a 65 Mb region flanked by markers D15Mit13 and rs31271969. It harbors 83 genes in highly polymorphic areas, 310 genes in all. Complete dissection of these loci will lead to identification of genetic variants that influence SZS in mice and provide a better understanding of seizure biology.

Monocytic cell type-specific transcriptional induction of collagenase.

Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences.

Amplification in human breast cancer of a gene encoding a c-myc mRNA-binding protein.

The coding region determinant-binding protein (CRD-BP) binds in vitro to c-myc mRNA and is thought to stabilize the mRNA and increase c-Myc protein abundance. The CRD-BP gene has 15 exons and 14 introns, is single-copy, and is located on chromosome 11 in mice and 17 in humans, close to HER-2/neu. The CRD-BP gene is moderately amplified in 12 of 40 human breast cancers; it is highly amplified in 2 others (14.4 and 20 copies). Despite their proximity, CRD-BP and HER-2/neu genes can be amplified independently. Amplification of a gene that might up-regulate c-Myc abundance could accelerate breast cancer.

Oxidative stress induces DNA damage and inhibits the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear leukocytes.

Human mononuclear leukocytes were exposed to prooxidants such as H2O2, phorbol-12-myristate-13-acetate, and 4-nitroquinoline-N-oxide, and the effects on induction of DNA damage and repair were evaluated. ADP ribosylation was activated by prooxidant exposure and the response was bimodal with peaks of activation occurring at about 30 min and 4-5 h. Other evidence for prooxidant-induced DNA damage was provided by nucleoid sedimentation assays. Unscheduled DNA synthesis (UDS) was only slightly induced by prooxidant exposure which suggested that either the DNA lesions were repaired by a short patch mechanism involving little UDS, or the repair process was inhibited by prooxidant exposures, or some combination of both. This point was clarified by the fact that the repair of DNA lesions induced by N-acetoxy-2-acetylaminofluorene, an inducer of large patch DNA repair, was inhibited in a dose-dependent manner by exposure to H2O2 and the inhibition was dependent on ADP ribosylation. In contrast, the repair of DNA strand breaks induced by prooxidant exposures as identified above were complete within about 8 h and the repair was independent of ADP ribosylation. Both ADP ribosylation and N-acetoxy-2-acetylaminofluorene-induced UDS were shown to be up- and down-regulated by the redox state of human mononuclear leukocytes indicating a unique mechanism of cellular control over DNA repair.

A delta-opioid receptor genetic variant is associated with abstinence prior to and during cocaine dependence treatment.

INTRODUCTION: An intronic polymorphism in the delta-opioid receptor gene (OPRD1) was previously associated with cocaine dependence in African-Americans. However, it is not known if the polymorphism (rs678849) is associated with dependence-related phenotypes within the cocaine dependent population. METHODS: Cocaine and alcohol dependent subjects were randomized to either topiramate or placebo. Abstinence from cocaine use was confirmed by urine drug screens for benzoylecgonine three times per week. Cocaine withdrawal and craving were assessed at randomization using the Cocaine Selective Severity Assessment (CSSA) and Minnesota Cocaine Craving Scale (MCCS), respectively. Subjects were also interviewed using the Addiction Severity Index (ASI). Genotype at rs678849 was determined for 105 African-American subjects and compared to cocaine abstinence, as well as scores for CSSA, MCCS, and ASI. RESULTS: African-American patients with the C/T or T/T genotypes (n=40) were more likely to be abstinent at the first urine drug screen and more likely to be abstinent for the week prior to randomization compared to patients with the C/C genotype (n=65). Subjects carrying the T allele were also more likely to have abstinent weeks over the course of the trial compared to those with the C/C genotype (RR=1.88, 95% CI=1.59-2.22, p=0.0035). No effects of rs678849 genotype on withdrawal, craving, or addiction severity were observed. CONCLUSIONS: A polymorphism in OPRD1 appears to be associated with both cocaine dependence and cocaine use during treatment in African-Americans. Follow-up studies to confirm the effect on cocaine use are warranted.

The accuracy of patient encounter logbooks used by family medicine clerkship students.

BACKGROUND: Family medicine predoctoral programs frequently have medical students record patient diagnoses in logbooks. Little is known about the accuracy of such logbooks. No studies have compared patient records dictated by students with cases recorded in logbooks. METHODS: Over 2 years, all patient encounters dictated by 79 medical students during their 8-week family medicine rotations were recorded and compared with information in the students' logbooks. RESULTS: Students dictated 2,520 patient encounters but only recorded 2,085 (82.7%) of them in their logbooks. Still, this rate of inclusion is higher than other studies where students did not dictate patient encounters. On the average, each student saw and dictated 32 patient encounters but omitted five to six from their logs. There were no significant differences between the 10 honors and 69 non-honors students in the proportion of patients omitted from logbooks. CONCLUSIONS: Medical students underreport patient encounters in clerkship logbooks. Keeping a record of the patients dictated by medical students was helpful in determining the accuracy of students' logbooks.

The Lesser Bushbaby (Galago senegalensis) and the Chacma baboon (Papio ursinus) as laboratory research animals.

The 2 species are evaluated as far as adaptibility, economics, reproductive performance and behaviour are concerned. An important limitation is availability which makes conservation and breeding programmes imperative.

Analysis of candidate genes for morphine preference quantitative trait locus Mop2.

Compared to DBA/2J (D2), C57BL/6J (B6) inbred mice exhibit strong morphine preference when tested using a two-bottle choice drinking paradigm. A morphine preference quantitative trait locus (QTL), Mop2, was originally mapped to proximal chromosome (Chr) 10 using a B6xD2 F2 intercross population, confirmed with reciprocal congenic strains and fine mapped with recombinant congenic strains. These efforts identified a ∼ 10-Million base pair (Mbp) interval, underlying Mop2, containing 35 genes. To further reduce the interval, mice from the D2.B6-Mop2-P1 congenic strain were backcrossed to parental D2 mice and two new recombinant strains of interest were generated: D2.B6-Mop2-P1.pD.dB and D2.B6-Mop2-P1.pD.dD. Results obtained from testing these strains in the two-bottle choice drinking paradigm suggest that the gene(s) responsible for the Mop2 QTL is one or more of 22 remaining within the newly defined interval (∼ 7.6 Mbp) which includes Oprm1 and several other genes related to opioid pharmacology. Real-time qRT-PCR analysis of Oprm1 and opioid-related genes Rgs17, Ppp1r14c, Vip, and Iyd revealed both between-strain and within-strain expression differences in comparisons of saline- and morphine-treated B6 and D2 mice. Analysis of Rgs17 protein levels also revealed both between-strain and within-strain differences in comparisons of saline- and morphine-treated B6 and D2 mice. Results suggest that the Mop2 QTL represents the combined influence of multiple genetic variants on morphine preference in these two strains. Relative contributions of each variant remain to be determined.

VMAT1 deletion causes neuronal loss in the hippocampus and neurocognitive deficits in spatial discrimination.

Vesicular monoamine transporters (VMAT) are involved in presynaptic storage and release of neurotransmitters. While it was thought initially that only VMAT2 is brain expressed and VMAT1 is present only in the periphery, recent data have challenged the exclusive expression of VMAT2 in the brain. To further elucidate the role of VMAT1 brain expression and its potential role in neuropsychiatric disorders, we have investigated mice lacking VMAT1. Comparison of wildtype and knock-out (KO) mice using qPCR and immunohistochemistry documents the expression of VMAT1 in the brain. Deletion of VMAT1 leads to increased hippocampal apoptosis and reduced neurogenesis as assessed by caspase-3-labeling and 5-bromo-deoxy-uridine-labeling. Behavioral data show that mice lacking VMAT1 have neurocognitive deficits. VMAT2 expression is not altered in VMAT1 KO mice, suggesting a distinct role of VMAT1. Our data support VMAT1 brain expression and suggest that VMAT1 plays a key role in survival of hippocampal neurons and thus might contribute to neurocognitive deficits observed in neuropsychiatric disorders.

Case-control association analysis of polymorphisms in the δ-opioid receptor, OPRD1, with cocaine and opioid addicted populations.

BACKGROUND: Addiction susceptibility and treatment responsiveness are greatly influenced by genetic factors. Sequence variation in genes involved in the mechanisms of drug action have the potential to influence addiction risk and treatment outcome. The opioid receptor system is involved in mediating the rewarding effects of cocaine and opioids. The µ-opioid receptor (MOR) has traditionally been considered the primary target for opioid addiction. The MOR, however, interacts with and is regulated by many known MOR interacting proteins (MORIPs), including the δ-opioid receptor (DOR). METHODS: The present study evaluated the contribution of OPRD1, the gene encoding the DOR, to the risk of addiction to opioids and cocaine. The association of OPRD1 polymorphisms with both opioid addiction (OA) and cocaine addiction (CA) was analyzed in African American (OA n=336, CA n=503) and European American (OA n=1007, CA n=336) populations. RESULTS: The primary finding of this study is an association of rs678849 with cocaine addiction in African Americans (allelic p=0.0086). For replication purposes, this SNP was analyzed in a larger independent population of cocaine addicted African Americans and controls and the association was confirmed (allelic p=4.53 × 10(-5); n=993). By performing a meta-analysis on the expanded populations, the statistical evidence for an association was substantially increased (allelic p=8.5 × 10(-7)) (p-values non-FDR corrected). CONCLUSION: The present study suggests that polymorphisms in OPRD1 are relevant for cocaine addiction in the African American population and provides additional support for a broad role for OPRD1 variants in drug dependence.

Quantitative trait loci for electrical seizure threshold mapped in C57BLKS/J and C57BL/10SnJ mice.

We mapped the quantitative trait loci (QTL) that contribute to the robust difference in maximal electroshock seizure threshold (MEST) between C57BLKS/J (BKS) and C57BL10S/J (B10S) mice. BKS, B10S, BKS × B10S F1 and BKS × B10S F2 intercross mice were tested for MEST at 8-9 weeks of age. Results of F2 testing showed that, in this cross, MEST is a continuously distributed trait determined by polygenic inheritance. Mice from the extremes of the trait distribution were genotyped using microarray technology. MEST correlated significantly with body weight and sex; however, because of the high correlation between these factors, the QTL mapping was conditioned on sex alone. A sequential series of statistical analyses was used to map QTLs including single-point, multipoint and multilocus methods. Two QTLs reached genome-wide levels of significance based upon an empirically determined permutation threshold: chromosome 6 (LOD = 6.0 at ∼69 cM) and chromosome 8 (LOD = 5.7 at ∼27 cM). Two additional QTLs were retained in a multilocus regression model: chromosome 3 (LOD = 2.1 at ∼68 cM) and chromosome 5 (LOD = 2.7 at ∼73 cM). Together the four QTLs explain one third of the total phenotypic variance in the mapping population. Lack of overlap between the major MEST QTLs mapped here in BKS and B10S mice and those mapped previously in C57BL/6J and DBA/2J mice (strains that are closely related to BKS and B10S) suggest that BKS and B10S represent a new polygenic mouse model for investigating susceptibility to seizures.

Effect of saccharin on growth and acid production of glucose-grown pathogenic and oral bacteria.

Growth and acid production of glucose-grown Gram-positive and Gram-negative rods as well as cocci from the human oral cavity were studied in the presence of 0.02 to 20.00 mg/ml sodium saccharin. All Gram-positive rods, i.e. Actinomyces viscosus, Lactobacillus acidophilus, Bacillus subtilis and Corynebacterium diphtheriae, and Gram-positive cocci, i.e. Streptococcus spp, Staphylococcus aureus and Micrococcus luteus, were significantly inhibited by saccharin, especially at the higher concentrations. While Gram-negative cocci, i.e. Veillonella sp and Neisseria sicca were strongly inhibited by all tested saccharin concentrations, Gram-negative rods, i.e. the enterics and Acinetobacter sp, exhibited little if any inhibition. Saccharin caused a significant reduction in fermentative acid production congruent with observed growth.

Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBP-beta site.

In this report, we demonstrate that the AP-1 site and a distal promoter element regulate transcriptional induction of collagenase-1 during monocytic differentiation. Chloramphenicol acetyltransferase expression constructs containing regions of the human collagenase-1 promoter were stably or transiently transfected into U937 cells, and reporter activity was assessed at various times after the onset of phorbol 12-myristate 13-acetate (PMA)-mediated differentiation. Rapid and strong induction of promoter activity was lost in constructs with a mutant AP-1 element; however, at 16-96 h post-PMA, the mutant collagenase-1 promoter displayed AP-1 independent PMA-mediated transactivation. The AP-1 mutant constructs also showed delayed transcriptional activation in PMA-treated fibroblasts. Western and supershift analyses indicated that functional Jun and Fos proteins were present in nuclear extracts of PMA-differentiated U937 cells. Promoter deletion constructs demonstrated the potential role of distal promoter sequences in regulating collagenase-1 transcription. In particular, Western, supershift, and promoter deletion analyses suggested a role for CCAAT/enhancer-binding protein-beta (C/EBP-beta) binding site between -2010 and -1954 in regulating transcription of collagenase-1 in monocytic cells. Our findings suggest that distinct regulatory elements, acting somewhat independently of each other, control expression of collagenase-1. In addition, our data suggests that the rapid PMA-mediated induction of collagenase-1 transcription is controlled by a mechanism distinct from that regulating the sustained expression of this proteinase in activated macrophages.

Hamster fibroblasts defective in thrombin-induced mitogenesis. A selection for mutants in phosphatidylinositol metabolism and other functions.

Growth of Chinese hamster lung fibroblasts (CCL39) on thrombin as sole mitogen is dependent on phosphatidylinositol (PI) metabolism and activation of the Na+/H+ antiporter. By modifying a H+ suicide selection developed for the isolation of antiporter mutants in these cells, we enriched for and isolated CCL39 variants deficient in the thrombin mitogenic response (thrombin nongrowers). These mutants retain alternate mitogenic mechanisms and, hence, grow well on media containing serum. When challenged with thrombin, the mutants show decreased, increased, or unchanged levels of inositol phosphates produced as compared with wild type cells. One of the mutants (D1-6b) has decreased inositol phosphates production not only with thrombin but also with serotonin (5-hydroxytryptamine) and AlF4-, suggesting a defect distal to the thrombin receptors. Extracts of this mutant reveal marked decreased phospholipase C activity toward PI. From the different phenotypes of the thrombin nongrowers, it is clear that the selection is general and that mutants with various biochemical defects should lead to a better understanding of the PI cycle as well as of functions essential to mitogenesis.