Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

Design and anticipated outcomes of the eMERGE-PGx project: a multicenter pilot for preemptive pharmacogenomics in electronic health record systems.

We describe here the design and initial implementation of the eMERGE-PGx project. eMERGE-PGx, a partnership of the Electronic Medical Records and Genomics Network and the Pharmacogenomics Research Network, has three objectives: (i) to deploy PGRNseq, a next-generation sequencing platform assessing sequence variation in 84 proposed pharmacogenes, in nearly 9,000 patients likely to be prescribed drugs of interest in a 1- to 3-year time frame across several clinical sites; (ii) to integrate well-established clinically validated pharmacogenetic genotypes into the electronic health record with associated clinical decision support and to assess process and clinical outcomes of implementation; and (iii) to develop a repository of pharmacogenetic variants of unknown significance linked to a repository of electronic health record-based clinical phenotype data for ongoing pharmacogenomics discovery. We describe site-specific project implementation and anticipated products, including genetic variant and phenotype data repositories, novel variant association studies, clinical decision support modules, clinical and process outcomes, approaches to managing incidental findings, and patient and clinician education methods.

Entry demographics and pharmacological treatment of migraine patients referred to a tertiary care pain clinic.

The aim was to examine pharmacological treatment of migraine patients admitted to a tertiary care pain clinic. A retrospective review of 100 consecutive migraine patients admitted to The Wasser Pain Management Centre was conducted. Patients included met the 2nd Edition of the International Classification of Headache Disorders criteria for diagnosis of migraine. Data were collected with regard to nicotine and alcohol consumption, family history of migraine headaches, other pain diagnoses and pharmacological treatment. Twenty-two per cent of these patients were male as opposed to 78% female. The mean age of patients admitted for migraine was 43.4 years. Of the patients admitted, 48% had tried at least one triptan in the past and only 31% were actively using triptan(s). The most commonly used triptan in the past had been sumatriptan, whereas the most common triptan used on admission was rizatriptan. Opiate use was much more prevalent; 72% of admitted patients were using an opiate and 27% used multiple opiates. A significant number of patients had not yet been tried on a triptan despite meeting the diagnostic criteria for migraine and having significant disability. More education of the general medical community may be beneficial in implementing a stratified care approach to migraine management.

Dopamine D2 receptor inhibition of adenylyl cyclase is abolished by acute ethanol but restored after chronic ethanol exposure (tolerance).

Dopamine D2 (D2) receptors seem to mediate reinforcing responses to addicting drugs. A stably transfected NG108-15 cell line expressing the long form of the rat brain D2 receptor (D2L) was used to determine how ethanol modifies D2 receptor coupling to adenylyl cyclase. Activation of D2L receptors by the D2 receptor-specific agonist R-(-)-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) inhibits both basal and receptor-stimulated cAMP production in these cells. Ethanol added acutely prevents D2L receptor inhibition of cAMP production. After chronic exposure to ethanol, however, D2L receptor coupling to adenylyl cyclase becomes tolerant to rechallenge with ethanol, i.e., ethanol no longer inhibits D2L receptor coupling and NPA inhibition of cAMP production is restored. Acute ethanol does not change NPA binding to D2 receptor in cell membranes but abolishes guanosine-5'-O-(3-thio)triphosphate induction of a lower-affinity state; chronic ethanol is without effect. The protein kinase A (PKA) inhibitor adenosine 3',5' cyclic monophosphorothioate, Rp-isomer, prevents acute ethanol inhibition of D2L receptor coupling. In contrast, the PKA activator adenosine 3',5' cyclic monophosphorothioate, Sp-isomer, reverses chronic ethanol-induced tolerance of D2L receptor coupling, restoring coupling to an ethanol-sensitive state. These results suggest that D2L receptor coupling to adenylyl cyclase via G(i) develops tolerance to ethanol inhibition, which appears to be influenced by PKA activity.

End-of-life issues.

In the Western world we have not fully accepted the fact that dying is indeed part of the process of living. Even today in the 21st century, physicians who have historically been taught to diagnose and treat disease with the expectation of cure, still regard death as a failure. We need to be reminded of the 15th-century definition of medical care, "To cure sometimes, to relieve often, to comfort always." We must realise that we cannot defy death and that every individual on earth has no choice but to travel the journey, be it long or short, from birth to ultimate death.

Cloning of a novel isoform of the mouse NBMPR-sensitive equilibrative nucleoside transporter (ENT1) lacking a putative phosphorylation site.

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [(3)H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.

Ethanol acts synergistically with a D2 dopamine agonist to cause translocation of protein kinase C.

Ethanol and other drugs of abuse increase synaptic dopamine levels; however, little is known about how ethanol alters dopaminergic signaling. We have reported that ethanol induces translocation of delta and epsilon protein kinase C (PKC) in neural cells in culture. Using NG108-15 and Chinese hamster ovary cell lines that express the dopamine D2 receptor (D2R), we show here that the D2R agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (NPA) also causes translocation of delta and epsilon PKC to the same sites as ethanol-induced translocation. D2R agonist and ethanol-induced translocation of delta and epsilon PKC share a common pathway that is blocked by pertussis toxin and requires phospholipase C (PLC) activity. These data suggest that both D2R agonists and ethanol activate PLC via a trimeric G protein leading to production of diacylglycerol with subsequent activation and translocation of delta and epsilon PKC. Moreover, ethanol and NPA, when present together at low concentrations that alone are ineffective, act synergistically to cause translocation of delta and epsilon PKC. Our data suggest that ethanol causes translocation of delta and epsilon PKC but cells expressing the D2R, such as neurons in the nucleus accumbens, may be particularly sensitive to low concentrations of ethanol.

Sensory and affective components of orofacial pain: is it all in your brain?

In this paper, we shall review several chronic orofacial pain conditions with emphasis on those that are essentially refractory to treatment. We shall present a review of current and past literature that describes the various pain phenomena as well as their underlying central mechanisms. New data concerning refractory pain will be used to underscore the importance of central processing of pain, with particular emphasis on neuropsychological and cognitive function and capacity that may play important roles in pain processing and maintenance of the pain state. Further, neurophysiological data showing that the anterior cingulate cortex (ACC) and other structures in the brain may play key roles in modulation of chronic pain will also be discussed. Although peripheral triggering events surely play an important role in initiating pain, the development of chronic and, in particular, refractory pain may depend on changes or malfunctions in the central nervous system. These changes may be quite subtle and require sophisticated approaches, such as functional MRI, to study them, as is now being done. New findings obtained therefore may lead to more rational and reliable treatment for orofacial pain.

Genomic organization and expression of the mouse equilibrative, nitrobenzylthioinosine-sensitive nucleoside transporter 1 (ENT1) gene.

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5'-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5'-flanking region showed that the nucleotide sequence from -652 to -111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (-296/-303, -307/-313) and two MAZ binding sites (-353/-359, -522/-528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

Ethanol-induced translocation of cAMP-dependent protein kinase to the nucleus. Mechanism and functional consequences.

Ethanol induces translocation of the catalytic subunit (Calpha) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108-15 cells. Ethanol also induces translocation of the RIIbeta regulatory subunit of PKA to the nucleus; RI and Cbeta are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Calpha with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Calpha by the PKA inhibitor PKI. Pre-incubation of Calpha with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Calpha is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R(p))-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Calpha and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

L-deprenyl in Alzheimer's disease: cognitive and behavioral effects.

BACKGROUND: Short-term studies of L-deprenyl in Alzheimer's disease (AD) suggest a beneficial effect, whereas longer-term studies are less convincing. Accordingly, we undertook a 6-month, randomized, double-blind, placebo-controlled clinical trial to assess the potential benefit of L-deprenyl in AD. METHODS: Sixty subjects were assigned to L-deprenyl (10 mg daily) or placebo. After 4 weeks of single-blind placebo, 51 subjects entered the double-blind phase. The Brief Psychiatric Rating Scale (BPRS) was the primary outcome measure. Secondary outcome measures were the Mini-Mental State Examination, Global Deterioration Scale, Alzheimer's Disease Assessment Scale (noncognitive), Cornell Scale for Depression in Dementia, Buschke Selective Reminding Test (BSRT), Relative's Assessment of Global Symptomatology-Elderly (RAGS-E), Controlled Oral Word Association Test, and Modified Continuous Performance Test. In addition, several exploratory tasks were included for future hypothesis testing. RESULTS: We found no significant differences between the L-deprenyl and placebo groups on the primary or secondary measures. However, several measures appeared to be sensitive to change over time, including the total score on the BPRS and some of its components as well as parts of the BSRT and the RAGS-E. CONCLUSION: Oral L-deprenyl provides no detectable benefit on general behavior, neuropsychiatric symptoms, or cognitive function in AD after 6 months of treatment. Protocols for future drug studies should utilize measures that are sensitive to change over time such as the BPRS.

Varying effects of ethanol on transfected cell lines.

The use of transfected cell lines has provided a powerful approach to study the functions of transfected proteins. This approach has also proven useful to determine the effects of acute and chronic ethanol exposure on particular proteins. We show here, however, that the effects of ethanol on transfected proteins vary between transiently transfected cells and stably transfected cell lines. Moreover, we found that the effect of ethanol on a particular protein depends on the plasmid vectors used for the transfection.

Anchoring proteins for protein kinase C: a means for isozyme selectivity.

Protein kinase C (PKC) isozymes comprise a family of related enzymes. There are only limited differences between these isozymes in substrate specificity or sensitivity to activators. However, there are multiple isozymes within a cell mediating isozyme-specific functions. Differential subcellular localization has been proposed to explain this specificity. When members of the PKC family are activated by lipid-derived second messengers, they translocate from one cell compartment to another. Isozyme specificity appears to be mediated in part by association of each PKC isozyme with specific anchoring proteins. This review will cover the proteins involved in the anchoring of PKC isozymes at specific subcellular sites, the domains in the PKC isozymes that mediate protein-protein interaction with isozyme-specific anchoring proteins, and identification of peptides that interfere with or promote these protein-protein interactions, thus altering the localization and function of individual isozymes.

Craniofacial pain: a multidisciplinary approach.

Patients with pain involving the head and orofacial complex are among the most challenging both for dentists and physicians. A collaborative approach to diagnosis and management will yield the best possible result for the patient and minimize aggravation and frustration for the clinician. This collaboration must include ongoing discussion between the various clinicians. Ultimately, the various practitioners will develop a deeper comprehension of each others' fields, and the boundaries of the various disciplines will become less definite and a truly transdisciplinary approach will result. The latter is a highly desirable outcome for clinicians and patients. (The patient presenting with an atypical facial pain has been used as an example of just such a situation and the brief dissertation above should not be interpreted as an in-depth discussion of this complex disorder.

Ethanol alters the subcellular localization of delta- and epsilon protein kinase C in NG108-15 cells.

Protein kinase C (PKC) has been shown to regulate the ethanol sensitivity of membrane-bound receptors and transporters, but little is known about the molecular mechanisms underlying this regulation. PKC is a family of isozymes that translocate to new intracellular sites on activation. Here we present immunochemical data showing that ethanol causes translocation of delta- and epsilon-PKC to new intracellular sites. Ethanol causes translocation of delta-PKC from the Golgi to the perinucleus; this translocation is similar to that induced by activation of PKC with phorbol esters. In contrast, epsilon-PKC translocation caused by ethanol is different from that induced by phorbol esters; ethanol causes translocation of epsilon-PKC from the perinucleus to the cytoplasm, whereas phorbol ester activation causes translocation of epsilon-PKC to the nucleus. Because the substrate specificity of these kinases is determined by their site of localization, ethanol-induced translocation of delta- and epsilon-PKC to new intracellular sites may explain some of the pleiotropic effects of ethanol on cellular functions.

A global perspective: technology in home care. Discussion.

Technological advances are changing the way the world shares information and conducts daily business, CARING asked home care colleagues around the world to respond to a question about technology's role in home care delivery. Although the degree of technology's involvement differs from country to country, each country is aware of the possibilities and is looking toward the future.

Cellular and molecular neuroscience of alcoholism.

Recent advances in neuroscience have made it possible to investigate the pathophysiology of alcoholism at a cellular and molecular level. Evidence indicates that ethanol affects hormone- and neurotransmitter-activated signal transduction, leading to short-term changes in regulation of cellular functions and long-term changes in gene expression. Such changes in the brain probably underlie many of the acute and chronic neurological events in alcoholism. In addition, genetic vulnerability also plays a role in alcoholism and, perhaps, in alcoholic medical disorders.