The taq IA and Ser311Cys polymorphisms in the dopamine D2 receptor gene and obesity

Dopamine D2 receptors (DRD2) in the central nervous system are involved in the regulation of feeding. It remains to be elucidated if mutations in the DRD2 gene contribute to the development of obesity. The aim of the present study was to investigate whether the Taq IA and Ser311Cys polymorphisms in the DRD2 gene are associated with obesity in Nauruan and Australian subjects. Subjects were selected based on extremes of the body mass index (BMI) distribution. Two groups of Australian women were selected. The leanest group had a mean BMI of 22.5 kg/m2 (range: 20.3-24.3) and the heaviest group had a mean of 36.1 kg/m2 (32.5-44.1). Four groups of Nauruan subjects were selected. Leanest men had a mean BMI of 33.0 kg/m2 (28.4-36.9), heaviest men had a mean of 52.8 kg/m2 (46.5-69.2), leanest women had a mean of 34.8 kg/m2 (28.2-41.8) and heaviest women had a mean of 55.1 kg/m2 (49.3-73.8). Subjects were genotyped for the Taq IA and Ser311Cys polymorphisms using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and allelic discrimination TaqmanTM PCR respectively. Leanest and heaviest groups were examined for differences in genotype frequency. Taq IA and Ser311Cys genotype frequencies did not differ significantly between leanest and heaviest Nauruan groups, or between leanest and heaviest Australians. Haplotype frequencies of these polymorphisms did not differ between leanest and heaviest groups. The Taq IA and Ser311Cys polymorphisms in the DRD2 gene are unlikely to be common causes of obesity in these populations.

Modeling gene-environment interaction in longitudinal data : risk for neuroticism due to interaction between maternal care and the Dopamine 4 Receptor gene (DRD4)

The purpose of this study was to investigate risk for neuroticism due to the joint action of low maternal care and compromised mesocorticolimbic ‘reward’ system function linked to a variable number tandem repeat (VNTR) in the dopamine 4 receptor gene (DRD4). Data were drawn from the Victorian Adolescent Health Cohort Study, a longitudinal study of the health and well-being of 2,000 young Australians followed from adolescence to young adulthood across 8 waves from 14- to 28-years. Genetic risk was defined by carriage of at least one copy of the 7-repeat allele or derivative alleles 5, 6, and 8 (labeled 7R+). Neuroticism was assessed in adolescence and young adulthood. We observed an approximately fourfold increase in the odds of reporting neurotic symptoms in carriers of the 7R+ disposition who reported low maternal care compared with non-carriers who reported high maternal care. The percentage of risk attributable to mechanisms in which both factors played a role was 35%. Findings are discussed in terms of implications for prevention.

Investigating interleukin-2 receptor family signaling in zebrafish

The zebrafish possesses all of the interleukin 2 receptor family except interleukin 2 receptor alpha and removal of the common signalling component interleukin 2 receptor gamma causes a human like severe combined immunodeficiency.

Wireless instantaneous neurotransmitter concentration system-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring - laboratory investigation

Object  In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time.

Methods  The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig.

Results   The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA.

Conclusions  By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

The prolactin response to sulpiride in major depression: the role of the D2 receptor in depression.

Multiple lines of investigations have implicated the role of the dopaminergic system in depression. The aim of the study was to characterise the Dopamine D2 receptor sensitivity status in depressed patients versus controls by means of a novel neuro-endocrine challenge test, the prolactin response to sulpiride. In this intervention, ten patients and ten age matched male volunteers were studied. The patients were diagnosed according to DSM-IV criteria, and Montgomery Asberg and Zung scales were done. There was no significant difference in baseline levels of prolactin between the depressed and control groups. Significantly higher prolactin levels after sulpiride challenge were however found in depressed patients than controls at all time points after sulpiride administration. This neuroendocrine challenge paradigm suggests that the prolactin response to sulpiride, a D2 receptor antagonist, is enhanced in depression, which suggests that this receptor might be supersensitive in depression compared to controls. This adds to the data implicating the dopaminergic system in the pathophysiology of depression, and suggests that dopaminergic mechanisms might be a target of therapeutic interest.

Identification of a gene for an ancient cytokine, interleukin 15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing binding motifs for IL-15Rα

Interleukins 2 and 15 (IL-2 and IL-15) are highly differentiated but related cytokines with overlapping, yet also distinct functions, and established benefits for medical drug use. The present study identified a gene for an ancient third IL-2/15 family member in reptiles and mammals, interleukin 15-like (IL-15L), which hitherto was only reported in fish. IL-15L genes with intact open reading frames (ORFs) and evidence of transcription, and a recent past of purifying selection, were found for cattle, horse, sheep, pig and rabbit. In human and mouse the IL-15L ORF is incapacitated. Although deduced IL-15L proteins share only ~21 % overall amino acid identity with IL-15, they share many of the IL-15 residues important for binding to receptor chain IL-15Rα, and recombinant bovine IL-15L was shown to interact with IL-15Rα indeed. Comparison of sequence motifs indicates that capacity for binding IL-15Rα is an ancestral characteristic of the IL-2/15/15L family, in accordance with a recent study which showed that in fish both IL-2 and IL-15 can bind IL-15Rα. Evidence reveals that the species lineage leading to mammals started out with three similar cytokines IL-2, IL-15 and IL-15L, and that later in evolution (1) IL-2 and IL-2Rα receptor chain acquired a new and specific binding mode and (2) IL-15L was lost in several but not all groups of mammals. The present study forms an important step forward in understanding this potent family of cytokines, and may help to improve future strategies for their application in veterinarian and human medicine.

Comorbidity between depression and inflammatory bowel disease explained by immune-inflammatory, oxidative, and nitrosative stress; tryptophan catabolite; and gut–brain pathways

The nature of depression has recently been reconceptualized, being conceived as the clinical expression of activated immune-inflammatory, oxidative, and nitrosative stress (IO&NS) pathways, including tryptophan catabolite (TRYCAT), autoimmune, and gut–brain pathways. IO&NS pathways are similarly integral to the pathogenesis of inflammatory bowel disease (IBD). The increased depression prevalence in IBD associates with a lower quality of life and increased morbidity in IBD, highlighting the role of depression in modulating the pathophysiology of IBD.This review covers data within such a wider conceptualization that better explains the heightened co-occurrence of IBD and depression. Common IO&NS underpinning between both disorders is evidenced by increased pro-inflammatory cytokine levels, eg, interleukin-1 (IL-1) and tumor necrosis factor-α, IL-6 trans-signalling; Th-1- and Th-17-like responses; neopterin and soluble IL-2 receptor levels; positive acute phase reactants (haptoglobin and C-reactive protein); lowered levels of negative acute phase reactants (albumin, transferrin, zinc) and anti-inflammatory cytokines (IL-10 and transforming growth factor-β); increased O&NS with damage to lipids, proteinsm and DNA; increased production of nitric oxide (NO) and inducible NO synthase; lowered plasma tryptophan but increased TRYCAT levels; autoimmune responses; and increased bacterial translocation. As such, heightened IO&NS processes in depression overlap with the biological underpinnings of IBD, potentially explaining their increased co-occurrence. This supports the perspective that there is a spectrum of IO&NS disorders that includes depression, both as an emergent comorbidity and as a contributor to IO&NS processes. Such a frame of reference has treatment implications for IBD when “comorbid” with depression.

Prevention of dabigatran-related gastrointestinal bleeding with gastroprotective agents: a population-based study

Background & Aims Use of dabigatran, an inhibitor of thrombin, increases the risk of gastrointestinal bleeding (GIB). However, it is not clear whether gastroprotective agents (GPAs) prevent GIB in dabigatran users. We investigated the risk of GIB and the role of gastroprotective agents (including proton pump inhibitors and histamine type-2-receptor antagonists) in patients using dabigatran. Methods We performed a retrospective cohort study using a population-wide database managed by the Hong Kong Hospital Authority. Patients newly prescribed dabigatran from 2010 through 2013 were included in the analysis. Poisson regression was used to assess the risk of GIB in dabigatran users by incidence rate ratio (IRR), adjusted for patient characteristics, comorbidities, and concurrent medications. Results Among the 5041 patients newly prescribed dabigatran, 124 (2.5%) developed GIB during follow-up evaluation (4.2/100 patient-years). The risk of GIB in this population increased among patients 75 years and older (IRR, 2.47; 95% confidence interval [CI], 1.66-3.68), patients with a history of peptic ulcers or GIB (IRR, 2.31; 95% CI, 1.54-3.46), and patients who used aspirin (IRR, 1.52; 95% CI, 1.03-2.24). Concomitant use of gastroprotective agents was associated with a reduced risk of GIB (IRR, 0.52; 95% CI, 0.35-0.77). Subcategory analysis showed that use of proton pump inhibitors (IRR, 0.53; 95% CI, 0.31-0.91) or histamine type-2-receptor antagonists (IRR, 0.61; 95% CI, 0.40-0.94) were associated with a lower risk of GIB. Further analysis showed that the risk reduction by gastroprotective agents was significant for only upper GIB (IRR, 0.29; 95% CI, 0.15-0.54), and only for patients with a prior history of peptic ulcers or GIB (IRR, 0.14; 95% CI, 0.06-0.30). Conclusions In the Hong Kong population, use of gastroprotective agents was associated with a reduced risk of GIB in patients taking dabigatran. The association was stronger for upper GIB than lower GIB, and in patients with a prior history of peptic ulcers or GIB.

Conserved IL-2Rγc signaling mediates lymphopoiesis in Zebrafish

The IL-2 receptor γ common (IL-2Rγc) chain is the shared subunit of the receptors for the IL-2 family of cytokines, which mediate signaling through JAK3 and various downstream pathways to regulate lymphopoiesis. Inactivating mutations in human IL-2Rγc result in SCID, a primary immunodeficiency characterized by greatly reduced numbers of lymphocytes. This study used bioinformatics, expression analysis, gene ablation, and specific pharmacologic inhibitors to investigate the function of two putative zebrafish IL-2Rγc paralogs, il-2rγc.a and il-2rγc.b, and downstream signaling components during early lymphopoiesis. Expression of il-2rγc.a commenced at 16 h post fertilization (hpf) and rose steadily from 4-6 d postfertilization (dpf) in the developing thymus, with il-2rγc.a expression also confirmed in adult T and B lymphocytes. Transcripts of il-2rγc.b were first observed from 8 hpf, but waned from 16 hpf before reaching maximal expression at 6 dpf, but this was not evident in the thymus. Knockdown of il-2rγc.a, but not il-2rγc.b, substantially reduced embryonic lymphopoiesis without affecting other aspects of hematopoiesis. Specific targeting of zebrafish Jak3 exerted a similar effect on lymphopoiesis, whereas ablation of zebrafish Stat5.1 and pharmacologic inhibition of PI3K and MEK also produced significant but smaller effects. Ablation of il-2rγc.a was further demonstrated to lead to an absence of mature T cells, but not B cells in juvenile fish. These results indicate that conserved IL-2Rγc signaling via JAK3 plays a key role during early zebrafish lymphopoiesis, which can be potentially targeted to generate a zebrafish model of human SCID.

Bias in emerging biomarkers for bipolar disorder

BACKGROUND: To date no comprehensive evaluation has appraised the likelihood of bias or the strength of the evidence of peripheral biomarkers for bipolar disorder (BD). Here we performed an umbrella review of meta-analyses of peripheral non-genetic biomarkers for BD. METHOD: The Pubmed/Medline, EMBASE and PsycInfo electronic databases were searched up to May 2015. Two independent authors conducted searches, examined references for eligibility, and extracted data. Meta-analyses in any language examining peripheral non-genetic biomarkers in participants with BD (across different mood states) compared to unaffected controls were included. RESULTS: Six references, which examined 13 biomarkers across 20 meta-analyses (5474 BD cases and 4823 healthy controls) met inclusion criteria. Evidence for excess of significance bias (i.e. bias favoring publication of 'positive' nominally significant results) was observed in 11 meta-analyses. Heterogeneity was high for (I 2 ⩾ 50%) 16 meta-analyses. Only two biomarkers met criteria for suggestive evidence namely the soluble IL-2 receptor and morning cortisol. The median power of included studies, using the effect size of the largest dataset as the plausible true effect size of each meta-analysis, was 15.3%. CONCLUSIONS: Our findings suggest that there is an excess of statistically significant results in the literature of peripheral biomarkers for BD. Selective publication of 'positive' results and selective reporting of outcomes are possible mechanisms.

Differential corticosteroid receptor regulation of mesoaccumbens dopamine efflux during the peak and nadir of the circadian rhythm : a molecular equilibrium in the midbrain?

Corticosteroid receptor modulation of mesoaccumbens dopamine neurotransmission is believed to be a key neurobiological mechanism mediating the effects of stress in addiction. Importantly, nucleus accumbens (NAc) subregions (core and shell) are reported to respond differentially to fluctuating basal levels of glucocorticoids, with dopaminergic responses in the core of the NAc being somewhat impervious to fluctuating levels of glucocorticoids relative to the shell. To investigate the corticosteroid receptor mechanisms mediating basal dopamine efflux in the core of the NAc, we have used chronoamperometry in combination with stearate-modified graphite paste electrodes in urethane anesthetized male Long–Evans rats during the peak and nadir of the circadian cycle. Blockade of ventral tegmental area low-affinity glucocorticoid (GR) or high-affinity mineralocorticoid (MR) receptors with mifepristone (1 μg/μl) or spironolactone (0.2 μg/μl), respectively, indicated that endogenous phase-dependent corticosteroid receptor activation (GRs during peak; MRs during nadir) facilitated extracellular NAc dopamine efflux. Conversely, the alternate receptor's actions appeared inhibitory at these time points (MRs during peak; GRs during nadir). Pharmacological activation of either the GR or MR with corticosterone (2 μg/μl) or aldosterone (0.2 μg/μl), respectively, potentiated NAc dopamine efflux, irrespective of circadian phase. Together, these data suggest that dominant corticosteroid receptor activation stimulates tonic mesoaccumbens dopamine transmission, enabling MRs and GRs to differentially maintain basal NAc dopamine release over the course of the circadian cycle. This points to an important molecular mechanism through which relatively stable NAc core dopamine extracellular levels could be maintained in the face of fluctuating corticosterone circadian rhythms.

New 2,N6-Disubstituted adenosines: Potent and selective A1 adenosine receptor agonists

A number of adenosine analogues substituted in the 2- and N⁶-positions were synthesized and evaluated for affinity, functional potency and intrinsic activity at the A₁ and A_2A adenosine receptors (AR). Three classes of N⁶-substituents were tested; norbornen-2-yl (series 1), norborn-2-yl (series 2) and 5,6-epoxynorborn-2-yl (series 3). The halogens; fluoro, bromo, and iodo were evaluated as C-2 substituents. All compounds showed relatively high affinity (nanomolar) for the A₁AR and high potency for inhibiting (−)isoproterenol-stimulated cAMP accumulation in hamster smooth muscle DDT1 MF-2 cells with the 2-fluoro derivatives from each series having the highest affinity. All of the derivatives showed the same intrinsic activity as CPA. At the A2AAR, all of the derivatives showed relatively low affinity and potency (micromolar) for stimulating cAMP accumulation in rat pheochromocytoma PC-12 cells. The intrinsic activity of the derivatives compared to CGS 21680 was dependent upon the halogen substituent in the C-2 position with most showing partial agonist activity. Of particular interest is 2-iodo-N⁶-(2S-endo-norborn-2-yl)adenosine (5e), which is over 100-fold selective for the A₁AR, is a full agonist at this receptor subtype and has no detectable agonist activity at the A_2AAR.

Insulin signaling after exercise in insulin receptor substrate-2-deficient mice

The period immediately after exercise is characterized by enhanced insulin action in skeletal muscle, and on the molecular level, by a marked increase in insulin-stimulated, phosphotyrosine-associated phosphatidylinositol (PI) 3-kinase activity. Because the increase in PI 3-kinase activity cannot be explained by increased insulin receptor substrate (IRS)-1 signaling, the present study examined whether this effect is mediated by enhanced IRS-2 signaling. In wild-type (WT) mice, insulin increased IRS-2 tyrosine phosphorylation (2.5-fold) and IRS-2-associated PI 3-kinase activity (3-fold). Treadmill exercise, per se, had no effect on IRS-2 signaling, but in the period immediately after exercise, there was a further increase in insulin-stimulated IRS-2 tyrosine phosphorylation (3.5-fold) and IRS-2-associated PI 3-kinase activity (5-fold). In IRS-2-deficient (IRS-2-/-) mice, the increase in insulin-stimulated, phosphotyrosine-associated PI 3-kinase activity was attenuated as compared with WT mice. However, in IRS-2-/- mice, the insulin-stimulated, phosphotyrosine-associated PI 3-kinase response after exercise was slightly higher than the insulin-stimulated response alone. In conclusion, IRS-2 tyrosine phosphorylation and associated PI 3-kinase activity are markedly enhanced by insulin in the immediate period after exercise. IRS-2 signaling can partially account for the increase in insulin-stimulated phosphotyrosine-associated PI 3-kinase activity after exercise.

Receptor activation and 2 distinct COOH- terminal motifs control G-CSF receptor distribution and internalization kinetics.

We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

Somatostatin modulates G-CSF-induced but not interleukin-3-induced proliferative responses in myeloid 32D cells via activation of somatostatin receptor subtype 2

Somatostatin, originally identified as a peptide involved in neurotransmission, functions as an inhibitor of multiple cellular responses, including hormonal secretion and proliferation. Somatostatin acts through activation of G-protein-coupled receptors of which five subtypes have been identified. We have recently established that human CD34/c-kit expressing hematopoietic progenitors and acute myeloid leukemia (AML) cells exclusively express SSTR2. A major mechanism implicated in the antiproliferative action of somatostatin involves activation of the SH2 domain-containing protein tyrosine phosphatase SHP-1. While 0.1-1 x 10(-9) M of somatostatin, or its synthetic stable analog octreotide, can inhibit G-CSF-induced proliferation of AML cells, little or no effects are seen on GM-CSF- or IL-3-induced responses.
MATERIALS AND METHODS: To study the mechanisms underlying the antiproliferative responses of myeloblasts to somatostatin, clones of the IL-3-dependent murine cell line 32D that stably express SSTR2 and G-CSF receptors were generated. RESULTS: Similar to AML cells, octreotide inhibited G-CSF-induced but not IL-3-induced proliferative responses of 32D[G-CSF-R/SSTR2] cells. Somatostatin induced SHP-1 activity and inhibited G-CSF-induced, but not IL-3-induced, activation of the signal transducer and activator of transcription proteins STAT3 and STAT5.
CONCLUSION: Based on these data and previous results, we propose a model in which recruitment and activation of the tyrosine phosphatase SHP-1 by SSTR2 is involved in the selective negative action of somatostatin on G-CSF-R signaling.