Alpha1b-adrenergic receptors control locomotor and rewarding effects of psychostimulants and opiates.

Drugs of abuse, such as psychostimulants and opiates, are generally considered as exerting their locomotor and rewarding effects through an increased dopaminergic transmission in the nucleus accumbens. Noradrenergic transmission may also be implicated because most psychostimulants increase norepinephrine (NE) release, and numerous studies have indicated interactions between noradrenergic and dopaminergic neurons through alpha1-adrenergic receptors. However, analysis of the effects of psychostimulants after either destruction of noradrenergic neurons or pharmacological blockade of alpha1-adrenergic receptors led to conflicting results. Here we show that the locomotor hyperactivities induced by d-amphetamine (1-3 mg/kg), cocaine (5-20 mg/kg), or morphine (5-10 mg/kg) in mice lacking the alpha1b subtype of adrenergic receptors were dramatically decreased when compared with wild-type littermates. Moreover, behavioral sensitizations induced by d-amphetamine (1-2 mg/kg), cocaine (5-15 mg/kg), or morphine (7.5 mg/kg) were also decreased in knock-out mice when compared with wild-type. Ruling out a neurological deficit in knock-out mice, both strains reacted similarly to novelty, to intraperitoneal saline, or to the administration of scopolamine (1 mg/kg), an anti-muscarinic agent. Finally, rewarding properties could not be observed in knock-out mice in an oral preference test (cocaine and morphine) and conditioned place preference (morphine) paradigm. Because catecholamine tissue levels, autoradiography of D1 and D2 dopaminergic receptors, and of dopamine reuptake sites and locomotor response to a D1 agonist showed that basal dopaminergic transmission was similar in knock-out and wild-type mice, our data indicate a critical role of alpha1b-adrenergic receptors and noradrenergic transmission in the vulnerability to addiction.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis.

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.

Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures.

INTRODUCTION: Breast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance. METHOD: To address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. RESULTS: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. CONCLUSION: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis.

Estrogen receptor-mediated signalling in female mice is locally activated in response to wounding.

Estrogen deprivation is associated with delayed healing, while Hormone Replacement Therapy (HRT) accelerates acute wound healing and protects against development of chronic wounds. Estrogen exerts its effects on healing via numerous cell types by signalling through the receptors ERα and ERβ, which bind to the Estrogen Responsive Element (ERE) and initiate gene transcription. The ERE-luciferase transgenic mouse model has been influential in assessing real-time in vivo estrogen receptor activation across a range of tissues and pathologies. Using this model we demonstrate novel temporally regulated peri-wound activation of estrogen signalling in female mice. Using histological methods we reveal that this signal is specifically localised to keratinocytes of the neoepidermis and wound margin dermal cells. Moreover using pharmacological agonists we reveal that ERβ induces ERE-mediated signal in both epidermal and dermal cells while ERα induces ERE-mediated signal in dermal cells alone. Collectively these novel data demonstrate rapid and regional activation of estrogen signalling in wounded skin. A more complete understanding of local hormonal signalling during repair is essential for the focussed development of new therapies for wound healing.

Estrogen receptor level determines sex-specific in vitro transcription from the Xenopus vitellogenin promoter.

Female-specific expression of the Xenopus laevis vitellogenin gene was reconstituted in vitro by addition of recombinant vaccinia-virus-produced estrogen receptor to nuclear extracts from male livers, in which this gene is silent. Transcription enhancement was at least 30 times and was selectively restricted to vitellogenin templates containing the estrogen-responsive unit. Thus, in male hepatocytes, estrogen receptor is the limiting regulatory factor that in the female liver controls efficient and accurate sex-specific expression of the vitellogenin gene. Furthermore, the Xenopus liver factor B, which is essential in addition to the estrogen receptor for the activation of this gene, was successfully replaced in the Xenopus extract by purified human nuclear factor I, identifying factor B of Xenopus as a functional homolog of this well-characterized human transcription factor.

Design, conduct, and analyses of Breast International Group (BIG) 1-98: a randomized, double-blind, phase-III study comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with receptor-positive, early breast cancer.

BACKGROUND: Aromatase inhibitors provide superior disease control when compared with tamoxifen as adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer. PURPOSE: To present the design, history, and analytic challenges of the Breast International Group (BIG) 1-98 trial: an international, multicenter, randomized, double-blind, phase-III study comparing the aromatase inhibitor letrozole with tamoxifen in this clinical setting. METHODS: From 1998-2003, BIG 1-98 enrolled 8028 women to receive monotherapy with either tamoxifen or letrozole for 5 years, or sequential therapy of 2 years of one agent followed by 3 years of the other. Randomization to one of four treatment groups permitted two complementary analyses to be conducted several years apart. The first, reported in 2005, provided a head-to-head comparison of letrozole versus tamoxifen. Statistical power was increased by an enriched design, which included patients who were assigned sequential treatments until the time of the treatment switch. The second, reported in late 2008, used a conditional landmark approach to test the hypothesis that switching endocrine agents at approximately 2 years from randomization for patients who are disease-free is superior to continuing with the original agent. RESULTS: The 2005 analysis showed the superiority of letrozole compared with tamoxifen. The patients who were assigned tamoxifen alone were unblinded and offered the opportunity to switch to letrozole. Results from other trials increased the clinical relevance about whether or not to start treatment with letrozole or tamoxifen, and analysis plans were expanded to evaluate sequential versus single-agent strategies from randomization. LIMITATIONS: Due to the unblinding of patients assigned tamoxifen alone, analysis of updated data will require ascertainment of the influence of selective crossover from tamoxifen to letrozole. CONCLUSIONS: BIG 1-98 is an example of an enriched design, involving complementary analyses addressing different questions several years apart, and subject to evolving analytic plans influenced by new data that emerge over time.

Role of MyD88 and Toll-like receptors 2 and 4 in the sensing of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88(-/-), TLR4(-/-), and TLR2(-/-) mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

Neuronal nicotinic receptors as new targets foramphetamine-induced oxidative damage and neurotoxicity

Amphetamine derivatives such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are drugs widely abused in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS) production seems to be one of the main causes. Recent research has demonstrated that blockade of 7 nicotinic acetylcholine receptors (nAChR) inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, 7 nAChR antagonists (methyllycaconitine and memantine) attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to 7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on 7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on 7 and heteromeric nAChR populations have been found.

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.

The effects of gamma-hydroxybutyrate, baclofen and GABAB receptors on brain activity and sleep in mice and humans

Currently, there is an increased interest in γ-hydroxybutyric acid (GHB) and its effects onsleep. This compound, sometimes referred to as 'rape drug', was recently approved as atreatment for the sleep disorder narcolepsy. Although several studies suggest that GHBinduces slow-wave sleep duration and improves sleep quality by increasing EEG slow-waveactivity, others question its ability to induce physiological sleep. GHB's mechanism of actionis still unclear, although in vivo and in vitro it seems to act at high doses as a low-affinityagonist of GABAB receptors. Furthermore, the role GABAB receptors play in sleep and theelectroencephalogram (EEG) is largely unknown.The aim of this project was therefore to investigate the effects of GHB on sleep and EEG, theinvolvement of GABAB receptors in mediating these effects, as well as the intrinsic role ofeach GABAB receptor subunit in the regulation of sleep. Thus, we administered GHB andbaclofen (BAC, a high-affinity agonist at GABAB receptor) to mice lacking the different GABABreceptor subunits and to healthy human volunteers.Our results, both in mice and humans, showed that GHB produced slow waves exclusivelythrough the stimulation of GABAB receptors, but did not induce physiological sleepnecessary to reduce sleep need and to increase cognitive performance. Unlike GHB, BACaffected the homeostatic regulation of sleep (sleep need) and induced a delayedhypersomnia. Finally, GABAB receptor and its subunits seem to play an important role insleep and in particular its circadian distribution.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

The peroxisome proliferator-activated receptors at the cross-road of diet and hormonal signalling.

The peroxisome proliferator-activated receptors (PPARs) are members of the steroid/thyroid nuclear receptor superfamily of ligand-activated transcription factors. To date, three isotypes have been identified, alpha, beta and gamma, encoded by three different genes. The alpha isotype is expressed at high levels in the liver where it has a role in lipid oxidation. Its expression and activity follow a diurnal rhythm that parallels the circulating levels of corticosterone in the bloodstream. The gamma isotype on the other hand, is mainly expressed in adipose tissue and has a critical role in adipocyte differentiation and lipid storage. The function of the ubiquitously expressed isotype, PPAR beta, remains to be determined. Besides fulfilling different roles in lipid metabolism, the different PPAR isotypes also have different ligand specificities. A new approach to identify ligands was developed based on the ligand-dependent interaction of PPAR with the recently characterized co-activator SRC-1. This so-called CARLA assay has allowed the identification of fatty acids and eicosanoids as PPAR ligands. Although the evidence clearly links PPAR isotypes to distinct functions, the molecular basis for this isotype-specificity is still unclear. All three isotypes are able to bind the same consensus response element, formed by a direct repeat of two AGGTCA hexamers separated by one base, though with different affinities. We recently demonstrated that besides the core DR-1 element, the 5' flanking sequence should be included in the definition of a PPRE. Interestingly, the presence of this flanking sequence is of particular importance in the context of PPAR alpha binding. Moreover, it reflects the polarity of the PPAR-RXR heterodimer on DNA, with PPAR binding to the 5' half-site and RXR binding to the 3' half-site. This unusual polarity may confer unique properties to the bound heterodimer with respect to ligand binding and interaction with co-activators and corepressors.