Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.

Avoiding transcription factor competition at promoter level increases the chances of obtaining oscillations

Background: The ultimate goal of synthetic biology is the conception and construction of genetic circuits that are reliable with respect to their designed function (e.g. oscillators, switches). This task remains still to be attained due to the inherent synergy of the biological building blocks and to an insufficient feedback between experiments and mathematical models. Nevertheless, the progress in these directions has been substantial. Results: It has been emphasized in the literature that the architecture of a genetic oscillator must include positive (activating) and negative (inhibiting) genetic interactions in order to yield robust oscillations. Our results point out that the oscillatory capacity is not only affected by the interaction polarity but by how it is implemented at promoter level. For a chosen oscillator architecture, we show by means of numerical simulations that the existence or lack of competition between activator and inhibitor at promoter level affects the probability of producing oscillations and also leaves characteristic fingerprints on the associated period/amplitude features. Conclusions: In comparison with non-competitive binding at promoters, competition drastically reduces the region of the parameters space characterized by oscillatory solutions. Moreover, while competition leads to pulse-like oscillations with long-tail distribution in period and amplitude for various parameters or noisy conditions, the non-competitive scenario shows a characteristic frequency and confined amplitude values. Our study also situates the competition mechanism in the context of existing genetic oscillators, with emphasis on the Atkinson oscillator.

CB1 knockout mice display impaired functionality of 5-HT1A and 5-HT2A/C receptors

Interaction between brain endocannabinoid (EC) and serotonin (5-HT) systems was investigated by examining 5-HT-dependent behavioural and biochemical responses in CB1 receptor knockout mice. CB1 knockout animals exhibited a significant reduction in the induction of head twitches and paw tremor by the 5-HT2A receptor selective agonist ()DOI, as well as a reduced hypothermic response following administration of the 5-HT1A receptor agonist (±)-8-OH-DPAT. Additionally, exposure to the tail suspension test induced enhanced despair responses in CB1 knockout mice. However, the tricyclic antidepressant imipramine and the 5-HT selective reuptake inhibitor fluoxetine induced similar decreases in the time of immobility in the tail suspension test in CB1 receptor knockout and wild-type mice. No differences were found between both genotypes with regard to 5-HT2A receptor and 5-HT1A receptors levels, measured by autoradiography in different brain areas. However, a significant decrease in the ability of the 5-HT1A receptor agonist (±)-8-OH-DPAT to stimulate 35SGTPS binding was detected in the hippocampal CA1 area of CB1 receptor knockout mice. This study provides evidence that CB1 receptors are involved in the regulation of serotonergic responses mediated by 5-HT2A and 5-HT1A receptors, and suggests that a reduced coupling of 5-HT1A receptors to Gi/o proteins in the hippocampus might be involved in these effects.

BDNF impairment in the hippocampus is related to enhanced despair behavior in CB1 knockout mice

Stress can cause damage and atrophy of neurons in the hippocampus by deregulating the expression of neurotrophic factors that promote neuronal plasticity. The endocannabinoid system represents a physiological substrate involved in neuroprotection at both cellular and emotional levels. The lack of CB1 receptor alters neuronal plasticity and originates an anxiety-like phenotype in mice. In the present study, CB1 knockout mice exhibited an augmented response to stress revealed by the increased despair behavior and corticosterone levels showed in the tail suspension test and decreased brain derived neurotrophic factor (BDNF) levels in the hippocampus. Interestingly, local administration of BDNF in the hippocampus reversed the increased despair behavior of CB1 knockout mice, confirming the crucial role played by BDNF on the emotional impairment of these mutants. The neurotrophic deficiency seems to be specific for BDNF since no differences were found in the levels of NGF and NT-3, two additional neurotrophic factors. Moreover, BDNF impairment is not related to the activity of its specific receptor TrkB or the activity of the transcription factor CREB. These results suggest that the lack of CB1 receptor originates an enhanced response to stress and neuronal plasticity by decreasing BDNF levels in the hippocampus that lead to impairment in the responses to emotional disturbances.

Attenuation of nicotine-induced antinociception, rewarding effects, and dependence in mu-opioid receptor knock-out mice

The involvement of μ-opioid receptors in different behavioral responses elicited by nicotine was explored by using μ-opioid receptor knock-out mice. The acute antinociceptive responses induced by nicotine in the tail-immersion and hot-plate tests were reduced in the mutant mice, whereas no difference between genotypes was observed in the locomotor responses. The rewarding effects induced by nicotine were then investigated using the conditioning place-preference paradigm. Nicotine produced rewarding responses in wild-type mice but failed to produce place preference in knock-out mice, indicating the inability of this drug to induce rewarding effects in the absence of μ-opioid receptors. Finally, the somatic expression of the nicotine withdrawal syndrome, precipitated in dependent mice by the injection of mecamylamine, was evaluated. Nicotine withdrawal was significantly attenuated in knock-out mutants when compared with wild-type mice. In summary, the present results show that μ-opioid receptors are involved in the rewarding responses induced by nicotine and participate in its antinociceptive responses and the expression of nicotine physical dependence.

Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.

Activity of the δ-Opioid Receptor Is Partially Reduced, Whereas Activity of the κ-Receptor Is Maintained in Mice Lacking the μ-Receptor

Previous pharmacological studies have indicated the possible existence of functional interactions between μ-, δ- and κ-opioid receptors in the CNS. We have investigated this issue using a genetic approach. Here we describe in vitro and in vivo functional activity of δ- and κ-opioid receptors in mice lacking the μ-opioid receptor (MOR). Measurements of agonist-induced [35S]GTPγS binding and adenylyl cyclase inhibition showed that functional coupling of δ- and κ-receptors to G-proteins is preserved in the brain of mutant mice. In the mouse vas deferens bioassay, deltorphin II and cyclic[d-penicillamine2,d-penicillamine5] enkephalin exhibited similar potency to inhibit smooth muscle contraction in both wild-type and MOR −/− mice. δ-Analgesia induced by deltorphin II was slightly diminished in mutant mice, when the tail flick test was used. Deltorphin II strongly reduced the respiratory frequency in wild-type mice but not in MOR −/− mice. Analgesic and respiratory responses produced by the selective κ-agonist U-50,488H were unchanged in MOR-deficient mice. In conclusion, the preservation of δ- and κ-receptor signaling properties in mice lacking μ-receptors provides no evidence for opioid receptor cross-talk at the cellular level. Intact antinociceptive and respiratory responses to the κ-agonist further suggest that the κ-receptor mainly acts independently from the μ-receptor in vivo. Reduced δ-analgesia and the absence of δ-respiratory depression in MOR-deficient mice together indicate that functional interactions may take place between μ-receptors and central δ-receptors in specific neuronal pathways.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta(2)-adrenergic receptor.

1. We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human beta(2) adrenergic receptor (beta(2)AR). 2. A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type beta(2)AR. 3. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. 4. We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the beta-ethanolamine tail with other residues of the beta(2)-AR binding pocket.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Effect of Pcbs (Aroclor 1254) on spermatogenesis In Testis Of Mouse

Polychlorinated biphenyls (PCBs) are a group of halogenated aromatic hydrocarbons, synthetic chemicals which do not occur naturally in the environment. PCBs are considered potential endocrine disruptors. They are estrogen-like and anti-androgenic chemicals in the environment contain potentially hazardous effects on male reproductive axis resulting in infertility and other hormonal dependent reproductive functions. These toxic substance cause alteration of the endocrine systems, mimic natural hormones and inhibit the action of hormones. The aim of this study is to examine the effect of Polychlorinated biphenyls (PCBs) on testicular development of male reproductive system in mice. The male mice were randomly assigned to five groups with each group comprising twenty-one members. In those mice were administered 0 μg/kg (control group) and 0.5, 5, 50, 500 μg/kg Aroclor 1254 (treated group) by gavages three time per week. Treatment was carried out for 50 days after which the mouse was sacrificed and the body weight, testicular weight; epedidymis weight, sperm mortality, sperm count and sperm abnormality were taken. However, there was no significant difference in testicular/body weight and epididymis/body weight ratio in treated group compared with the control group. According to the analysis of sperm quality, Aroclor 1254 treated group demonstrated significant increased in sperm mortality in 500 μg/kg; decreased the sperm count in 0.5 μg/kg, 5 μg/kg, 50 μg/kg and 500 μg/kg; and significantly elevate the sperm abnormality in 50 μg/kg and 500 μg/kg compared to the control in a dose-dependent manner. The sex hormone levels in the testes were detected by radio-immunoassay (RIA) method. The levels of testosterone and 17β-estradiol did not reveal significant alteration (p< 0.05) in PCBs treated groups compared to the control in a dose-dependent manner. The testis were obtained and subjected to routine histopathology following exposure to PCBs in supplement diet. The diameter of the seminiferous tubule and the number of Sertoli cells in the treated group increased significantly (p< 0.05) in comparison to the control group. For the spermatogenic cell, the number of germ cell in high concentration decreased significantly (p< 0.05). However, spermatogonia cells in PCB treated group showed non-significant difference (p< 0.05) compared to the control. vii Western blot analysis was used to determine the level of protein between the control and treated group. The level of Proliferating cell nuclear antigen (PCNA) was determined and the results have shown no significant alteration between the treated groups and the control. the level of sex hormone receptor (ER α/β); Androgen receptor (AR) were identified in the testes to detect the proliferative effect induced by PCBs. Statistical analyses of AR, ER α and ER β did not reveal significant difference between the control and the treated groups. In the present study, we continue to investigate adverse effect of Aroclor 1254 and their mechanism on spermatogenesis. The result of Sperm quality and histopathology showed that Aroclor 1254 at low concentration induce inhibitory effect on testicular function of male mouse.

Ezrin directly interacts with the alpha1b-adrenergic receptor and plays a role in receptor recycling.

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.

Making sense of AMPA receptor trafficking by modeling molecular mechanisms of synaptic plasticity.

Synaptic plasticity involves a complex molecular machinery with various protein interactions but it is not yet clear how its components give rise to the different aspects of synaptic plasticity. Here we ask whether it is possible to mathematically model synaptic plasticity by making use of known substances only. We present a model of a multistable biochemical reaction system and use it to simulate the plasticity of synaptic transmission in long-term potentiation (LTP) or long-term depression (LTD) after repeated excitation of the synapse. According to our model, we can distinguish between two phases: first, a "viscosity" phase after the first excitation, the effects of which like the activation of NMDA receptors and CaMKII fade out in the absence of further excitations. Second, a "plasticity" phase actuated by an identical subsequent excitation that follows after a short time interval and causes the temporarily altered concentrations of AMPA subunits in the postsynaptic membrane to be stabilized. We show that positive feedback is the crucial element in the core chemical reaction, i.e. the activation of the short-tail AMPA subunit by NEM-sensitive factor, which allows generating multiple stable equilibria. Three stable equilibria are related to LTP, LTD and a third unfixed state called ACTIVE. Our mathematical approach shows that modeling synaptic multistability is possible by making use of known substances like NMDA and AMPA receptors, NEM-sensitive factor, glutamate, CaMKII and brain-derived neurotrophic factor. Furthermore, we could show that the heteromeric combination of short- and long-tail AMPA receptor subunits fulfills the function of a memory tag.

Strong minimax lower bounds for learning

Minimax lower bounds for concept learning state, for example, thatfor each sample size $n$ and learning rule $g_n$, there exists a distributionof the observation $X$ and a concept $C$ to be learnt such that the expectederror of $g_n$ is at least a constant times $V/n$, where $V$ is the VC dimensionof the concept class. However, these bounds do not tell anything about therate of decrease of the error for a {\sl fixed} distribution--concept pair.\\In this paper we investigate minimax lower bounds in such a--stronger--sense.We show that for several natural $k$--parameter concept classes, includingthe class of linear halfspaces, the class of balls, the class of polyhedrawith a certain number of faces, and a class of neural networks, for any{\sl sequence} of learning rules $\{g_n\}$, there exists a fixed distributionof $X$ and a fixed concept $C$ such that the expected error is larger thana constant times $k/n$ for {\sl infinitely many n}. We also obtain suchstrong minimax lower bounds for the tail distribution of the probabilityof error, which extend the corresponding minimax lower bounds.

A model of shadow banking

We present a model of shadow banking in which financial intermediaries originate and trade loans, assemble these loans into diversified portfolios, and then finance these portfolios externally with riskless debt. In this model: i) outside investor wealth drives the demand for riskless debt and indirectly for securitization, ii) intermediary assets and leverage move together as in Adrian and Shin (2010), and iii) intermediaries increase their exposure to systematic risk as they reduce their idiosyncratic risk through diversification, as in Acharya, Schnabl, and Suarez (2010). Under rational expectations, the shadow banking system is stable and improves welfare. When investors and intermediaries neglect tail risks, however, the expansion of risky lending and the concentration of risks in the intermediaries create financial fragility and fluctuations in liquidity over time.