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We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.

The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca(2+) and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6(-/-) mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus.

The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg(2+) channels participate in transcellular Mg(2+) transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg(2+) reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na(+)/Cl(-) cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg(2+) reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal--and low--Mg(2+) diet. Increased urinary Mg(2+) wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg(2+) concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg(2+) diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels., (Copyright © 2016 by the American Society of Nephrology.)

Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates., Competing Interests: The author declares that no competing interests exist.

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.

Transendothelial migration (TEM) of leukocytes is the step in leukocyte emigration in which the leukocyte actually leaves the blood vessel to carry out its role in the inflammatory response. It is therefore, arguably the most critical step in emigration. This review focuses on two of the many aspects of this process that have seen important recent developments. The adhesion molecules, PECAM (CD31) and CD99 that regulate two major steps in TEM, do so by regulating specific signals. PECAM initiates the signaling pathway responsible for the calcium flux that is required for TEM. Calcium enters through the cation channel TRPC6 and recruits the first wave of trafficking of membrane from the lateral border recycling compartment (LBRC). CD99 signals through soluble adenylate cyclase to activate protein kinase A to recruit a second wave of LBRC trafficking. Another process that is critical for TEM is transient removal of VE-cadherin from the site of TEM. However, the local signaling pathways that are responsible for this appear to be different from those that open the junctions to increase vascular permeability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Recent studies have demonstrated that adenylate cyclase 3 (AC3) has a protective role in obesity. This gene resides at the pathway with glucagon-like peptide (GLP)-1. Liraglutide is a GLP-1 analog and has independent glucose and body weight (BW)-reducing effects. In the present study, we aimed to examine whether hepatic AC3 activity was regulated by Liraglutide and to further understand the effect of AC3 in reduction of BW and insulin resistance., Subjects: The diabesity and obese mice were induced from db/db and C57BL/6 J mice, respectively, by high-fat diet. Liraglutide (0.1 mg kg(-1) per 12 h) was given to the mice twice daily for 12 weeks. C57BL/6 J mice fed with chow diet and obese or diabesity mice treated with saline were used as the controls. Hepatic AC3 gene expression at mRNA and protein levels was analyzed with real-time reverse transcription-PCR and western blot. Fasting blood glucose and serum insulin levels were measured and followed insulin resistance index (HOMA-IR) was evaluated according to the homeostasis model assessment., Results: After administration of Liraglutide, BW and HOMA-IR in obese and diabesity mice were decreased, whereas hepatic AC3 mRNA and protein expression levels were upregulated. The AC3 gene expression was negatively correlated with BW, HOMA-IR and the area ratio of hepatic fat deposition in the liver., Conclusions: The present study thus provides the evidence that hepatic AC3 gene expression is upregulated by Liraglutide. The reduction of BW and improvement of insulin resistance with Liraglutide may be partially explained by AC3 activation.

Objective: Adenylyl cyclases (ACs) catalyze the synthesis of cAMP from ATP, and cAMP signaling affects a large number of neuronal processes. Ca(2+)-stimulated adenylyl cyclase 8 (AC8) expressed in the CNS plays a role in synaptic plasticity, drug addiction and ethanol sensitivity, and chronic pain. This study was to aim at examining the contributions of AC8 to epileptogenesis., Methods: In this study, we observed the seizure behavior induced by kainic acid (20 mg/kg or 30 mg/kg) or pilocarpine (350 mg/kg) in AC8 KO and wild-type mice. Next we injected kainic acid or pilocarpine to induce status epilepticus (SE), and examined neuronal degeneration (by Fluoro-Jade B staining) and mossy fiber sprouting (by Timm staining) 24h and 2 weeks after SE termination in the hippocampus, respectively. Finally, 15 min after intraperitoneal injection of kainic acid (30 mg/kg), we examined phosphor-ERK1/2 in the hippocampus by Western blot and immunochemistry staining., Results: We first observed that AC8 KO mutants display reduced susceptibility (including seizure latency and episodes) to two chemoconvulsants, kainic acid and pilocarpine. Moreover, we found that degenerative neurons and mossy fiber sprouting induced by chemoconvulsants were significant decreased in the hippocampus. Further, Western blot and immunochemistry analysis revealed that the MAPK signaling in the hippocampus was attenuated in kainic acid-injected AC8 KO mice., Conclusion: AC8 is involved in epileptogenesis, and may serve as a potential target for the treatment of epilepsy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The replicative DNA polymerases insert ribonucleotides into DNA at a frequency of approximately 1/6500 nucleotides replicated. The rNMP residues make the DNA backbone more susceptible to hydrolysis and can also distort the helix, impeding the transcription and replication machineries. rNMPs in DNA are efficiently removed by RNaseH2 by a process called ribonucleotides excision repair (RER). In the absence of functional RNaseH2, rNMPs are subject to cleavage by Topoisomerase I, followed by further processing to result in deletion mutations due to slippage in simple DNA repeats. The topoisomerase I-mediated cleavage at rNMPs results in DNA ends that cannot be ligated by DNA ligase I, a 5'OH end and a 2'-3' cyclic phosphate end. In the budding yeast, the mutation level in RNaseH2 deficient cells is kept low via the action of the Srs2 helicase and the Exo1 nuclease, which collaborate to process the Top1-induced nick with subsequent non-mutagenic gap filling. We have surveyed other helicases and nucleases for a possible role in reducing mutagenesis at Top1 nicks at rNMPs and have uncovered a novel role for the RecQ family helicase Sgs1 in this process.

The revue deals with the role of each component of adenylate cyclase regulatory system in the rat myometrial contractile activity modulation by the peptidoglycane of Staphylococcus aureus. Noradrenalin and salbutamol were used to investigate peptidoglycane impact on the myometrial β-adrenergic receptors. It was shown that inhibited by these substances myometrial contractility increased to the initial level after peptidoglycane application. The same effect we observed under the cAMP level elevation by forscolin. Peptidoglycan’ s ability to strengthen contractions was inhibited by the 8-brom-cAMP and papaverine application. Stimulation of Gs-protein by the cholera toxin didn’t influence on the peptidoglycane effect while the blocking of Gi/o-protein by the pertussis toxin caused stopping it’s manifestation. We concluded that the modulating effect of peptidoglycane implemented via Gi/o-protein activation, which causes adenilatcyclase desensitization.

Objective: To investigate the clinical spectrum and distinguishing features of adenylate cyclase 5 (ADCY5)-related dyskinesia and genotype-phenotype relationship., Methods: We analyzed ADCY5 in patients with choreiform or dystonic movements by exome or targeted sequencing. Suspected mosaicism was confirmed by allele-specific amplification. We evaluated clinical features in our 50 new and previously reported cases., Results: We identified 3 new families and 12 new sporadic cases with ADCY5 mutations. These mutations cause a mixed hyperkinetic disorder that includes dystonia, chorea, and myoclonus, often with facial involvement. The movements are sometimes painful and show episodic worsening on a fluctuating background. Many patients have axial hypotonia. In 2 unrelated families, a p.A726T mutation in the first cytoplasmic domain (C1) causes a relatively mild disorder of prominent facial and hand dystonia and chorea. Mutations p.R418W or p.R418Q in C1, de novo in 13 individuals and inherited in 1, produce a moderate to severe disorder with axial hypotonia, limb hypertonia, paroxysmal nocturnal or diurnal dyskinesia, chorea, myoclonus, and intermittent facial dyskinesia. Somatic mosaicism is usually associated with a less severe phenotype. In one family, a p.M1029K mutation in the C2 domain causes severe dystonia, hypotonia, and chorea. The progenitor, whose childhood-onset episodic movement disorder almost disappeared in adulthood, was mosaic for the mutation., Conclusions: ADCY5-related dyskinesia is a childhood-onset disorder with a wide range of hyperkinetic abnormal movements. Genotype-specific correlations and mosaicism play important roles in the phenotypic variability. Recurrent mutations suggest particular functional importance of residues 418 and 726 in disease pathogenesis., (© 2015 American Academy of Neurology.)

The cAMP/protein kinase A pathway regulates methamphetamine (METH)-induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild-type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH-induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)-evoked accumbal dialysate dopamine levels were similar between genotypes, saline-treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH-sensitized DKO mice compared to saline-treated controls, an effect not observed in WT mice. Notably, saline-treated DKO mice had significantly increased phosphorylated Dopamine- and cAMP-regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug-induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin-stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine-induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP-32 activation. These findings indicate AC 1/8 signaling regulates the sensitivity of the nigrostriatal pathway subserving stimulant and neuroadaptive sensitizing effects of methamphetamine. 3-MT, 3-methoxytyramine; Ca(2+), calcium; CaM, calmodulin; cdk5; cyclin-dependent kinase 5; DA, dopamine; DARPP-32, dopamine- and cAMP-regulated phosphoprotein; D1R, dopamine D1 receptor; HVA, homovanillic acid; PKA, protein kinase A., (© 2015 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of The International Society for Neurochemistry.)

The photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva was synthesized and the purified recombinant protein was characterized by biochemical and optical spectroscopic methods. TpPAC consists of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and an adenylyl cyclase homology domain (CHD). A light induced cAMP cyclase activity of ≈ 53.3 nmolmg(-1)min(-1) was measured while in the dark the cyclase activity was approximately a factor of 240 lower. The photo-cycling dynamics of the BLUF domain of TpPAC was studied by absorption spectra, fluorescence quantum distribution, and fluorescence lifetime measurements. The quantum efficiency of BLUF domain signaling state formation was found to be ϕs ≈ 0.59. A three-component exponential recovery of the signaling state to the receptor state was observed with the time constants τrec,1 = 4.8s, τrec,2 = 34.2s, and τrec,3 = 293s at 21.3 °C. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent TpPAC melting temperature of ϑm ≈ 46 °C was determined. The photo-degradation of TpPAC in the signaling state was studied by prolonged intense light exposure at 455 nm. An irreversible flavin photo-degradation was observed with quantum yield ϕD ≈ 8.7 × 10(-6)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The chicken pineal gland synthesizes and releases melatonin rhythmically in light/dark (LD) cycles, with high melatonin levels during the dark phase, and in constant darkness (DD) for several cycles before it gradually damps to arrhythmicity in DD. Daily administration of norepinephrine (NE) in vivo and in vitro prevents the damping and restores the melatonin rhythm. To investigate the role of the circadian clock on melatonin rhythm damping and of its restoration by NE, the effects of NE administration at different phases of the melatonin cycle revealed a robust rhythm in NE sensitivity in which NE efficacy in increasing melatonin amplitude peaked in late subjective night and early subjective day, suggesting a clock underlying NE sensitivity. However, NE itself had no effect on circadian phase or period of the melatonin rhythms. Transcriptional analyses indicated that even though the rhythm of melatonin output damped to arrhythmicity, messenger RNA (mRNA) encoding clock genes gper2, gper3, gBmal1, gclock, gcry1, and gcry2; enzymes associated with melatonin biosynthesis; and enzymes involved in cyclic nucleotide signaling remained robustly rhythmic. Of these, only gADCY1 (adenylate cyclase 1) and gPDE4D (cAMP-specific 3',5'-cyclic phosphodiesterase 4D) were affected by NE administration at the mRNA levels, and only ADCY1 was affected at the protein level. The data strongly suggest that damping of the melatonin rhythm in the chick pineal gland occurs at the posttranscriptional level and that a major role of the clock is to regulate pinealocytes' sensitivity to neuronal input from the brain., (© 2015 The Author(s).)

The most important physiological mechanism mediating enhanced exercise performance is increased sympathetic, beta adrenergic receptor (β-AR), and adenylyl cyclase (AC) activity. This is the first report of decreased AC activity mediating increased exercise performance. We demonstrated that AC5 disruption, that is, knock out (KO) mice, a longevity model, increases exercise performance. Importantly for its relation to longevity, exercise was also improved in old AC5 KO. The mechanism resided in skeletal muscle rather than in the heart, as confirmed by cardiac- and skeletal muscle-specific AC5 KO's, where exercise performance was no longer improved by the cardiac-specific AC5 KO, but was by the skeletal muscle-specific AC5 KO, and there was no difference in cardiac output during exercise in AC5 KO vs. WT. Mitochondrial biogenesis was a major mechanism mediating the enhanced exercise. SIRT1, FoxO3a, MEK, and the anti-oxidant, MnSOD were upregulated in AC5 KO mice. The improved exercise in the AC5 KO was blocked with either a SIRT1 inhibitor, MEK inhibitor, or by mating the AC5 KO with MnSOD hetero KO mice, confirming the role of SIRT1, MEK, and oxidative stress mechanisms. The Caenorhabditis elegans worm AC5 ortholog, acy-3 by RNAi, also improved fitness, mitochondrial function, antioxidant defense, and lifespan, attesting to the evolutionary conservation of this pathway. Thus, decreasing sympathetic signaling through loss of AC5 is not only a mechanism to improve exercise performance, but is also a mechanism to improve healthful aging, as exercise also protects against diabetes, obesity, and cardiovascular disease, which all limit healthful aging., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

It's known that blood leptin level is reduced in intrauterine growth retardation (IUGR) fetus, and placental leptin is the major source of fetal blood leptin. This study aimed to investigate the decreased fetal blood leptin level by prenatal caffeine exposure (PCE) and its underlying placental mechanisms. Pregnant Wistar rats were intragastrically administered caffeine (30-120 mg/kg day) from gestational day 9 to 20. The level of fetal serum leptin and the expression of placental leptin-related genes were analyzed. Furthermore, we investigated the molecular mechanism of the reduced placental leptin's expression by treatment with caffeine (0.8-20 μM) in the BeWo cells. In vivo, PCE significantly decreased fetal serum leptin level in caffeine dose-dependent manner. Meanwhile, placental mRNA expression of adenosine A2a receptor (Adora2a), cAMP-response element binding protein (CREB), a short-type leptin receptor (Ob-Ra) and leptin was reduced in the PCE groups. In vitro, caffeine significantly decreased the mRNA expression of leptin, CREB and ADORA2A in concentration and time-dependent manners. The addition of ADORA2A agonist or adenylyl cyclase (AC) agonist reversed the inhibition of leptin expression induced by caffeine. PCE induced a lower level of fetal blood leptin, which the primary mechanism is that caffeine inhibited antagonized Adora2a and AC activities to decreased cAMP synthesis, thus inhibited the expression of the transcription factor CREB and target gene leptin in the placenta. Meantime, the reduced transportation of maternal leptin by placental Ob-Ra also contributed to the reduced fetal blood leptin. Together, PCE decreased fetal blood leptin mainly via reducing the expression and transportation of leptin in the placenta., (Copyright © 2015 Elsevier Inc. All rights reserved.)

YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1-10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies have been an important model system for studying sparse coding in the olfactory system, where this format is important for accurate memory formation. In Drosophila, it has recently been shown that the 2,000 Kenyon cells of the mushroom body converge onto a population of only 34 mushroom body output neurons (MBONs), which fall into 21 anatomically distinct cell types. Here we provide the first, to our knowledge, comprehensive view of olfactory representations at the fourth layer of the circuit, where we find a clear transition in the principles of sensory coding. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit. Instead, at the population level, odour representations are reformatted so that positive and negative correlations arise between representations of different odours. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals, but that tuning of the same neuron across the two hemispheres of an individual fly was nearly identical. Thus, individualized coordination of tuning arises at this level of the olfactory circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to flexibly map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs. The reformatting of sensory representations we observe here may mark the beginning of this sensory-motor transition in the olfactory system.

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those in weakly stimulated regions. Surprisingly, reduction in neuronal excitability or ablation of the olfactory G protein in OSNs does not alter the cilia length pattern, indicating that neither spontaneous nor odor-evoked activity is required for its establishment. Furthermore, the pattern is evident at birth, maintained into adulthood, and restored following pharmacologically induced degeneration of the olfactory epithelium, suggesting that it is intrinsically programmed. Intriguingly, type III adenylyl cyclase (ACIII), a key protein in olfactory signal transduction and ubiquitous marker for primary cilia, exhibits location-dependent gene expression levels, and genetic ablation of ACIII dramatically alters the cilia pattern. These findings reveal an intrinsically programmed configuration in the nose to ensure high sensitivity to odors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) is the most common adult acute leukemia. Despite treatment, the majority of the AML patients relapse within 5 years. In silico analysis of several available databases of AML patients showed that the expression of adenylate cyclase 7 (ADCY7) significantly inversely correlates with the overall survival of AML patients. To determine whether ADCY7 supports AML development, we employed an shRNA-encoding lentivirus system to inhibit adcy7 expression in human AML cells including U937, MV4-11, and THP-1 cells. The ADCY7 deficiency resulted in decreased cell growth, elevated apoptosis, and lower c-Myc expression of these leukemia cells. This indicates that G protein-coupled receptor signaling contributes to AML pathogenesis. Our study suggests that inhibition of ADCY7 may be novel strategy for treating leukemia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on β-adrenoceptor (β-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to β-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins., (© FASEB.)

The role of cAMP- and IKK-2-dependent pathways in stimulation of the growth capacity of mesenchymal progenitor cells with alkaloid songorine was studied in vitro. Inhibitors of adenylate cyclase and IKK-2 were shown to abolish the increase in proliferative activity of progenitor cells. Moreover, blockade of the inhibitory kinase complex was accompanied by a decrease in the intensity of progenitor cell differentiation.

We investigated the role of Drosophila larva olfactory system in identification of congeners and aliens. We discuss the importance of these activities in larva navigation across substrates, and the implications for allocation of space and food among species of similar ecologies. Wild type larvae of cosmopolitan D. melanogaster and endemic D. pavani, which cohabit the same breeding sites, used species-specific volatiles to identify conspecifics and aliens moving toward larvae of their species. D. gaucha larvae, a sibling species of D. pavani that is ecologically isolated from D. melanogaster, did not respond to melanogaster odor cues. Similar to D. pavani larvae, the navigation of pavani female x gaucha male hybrids was influenced by conspecific and alien odors, whereas gaucha female x pavani male hybrid larvae exhibited behavior similar to the D. gaucha parent. The two sibling species exhibited substantial evolutionary divergence in processing the odor inputs necessary to identify conspecifics. Orco (Or83b) mutant larvae of D. melanogaster, which exhibit a loss of sense of smell, did not distinguish conspecific from alien larvae, instead moving across the substrate. Syn97CS and rut larvae of D. melanogaster, which are unable to learn but can smell, moved across the substrate as well. The Orco (Or83b), Syn97CS and rut loci are necessary to orient navigation by D. melanogaster larvae. Individuals of the Trana strain of D. melanogaster did not respond to conspecific and alien larval volatiles and therefore navigated randomly across the substrate. By contrast, larvae of the Til-Til strain used larval volatiles to orient their movement. Natural populations of D. melanogaster may exhibit differences in identification of conspecific and alien larvae. Larval locomotion was not affected by the volatiles.

The type I parathyroid hormone receptor (PTH1R) mediates PTH and PTH-related protein (PTHrP) actions on extracellular mineral ion homeostasis and bone remodeling. These effects depend in part on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Sequences located within or at the carboxyl-terminus of PTH1R control its activation and trafficking. β-catenin regulates PTH1R signaling and promotes chondrocyte hypertrophy through binding to the intracellular carboxyl-terminal region of the receptor. How the interaction of PTH1R with β-catenin affects PTH-stimulated ERK1/2 is unknown. In the present study, human embryonic kidney 293 (HEK293) cells, which do not express the PTH1R, were used to investigate whether the disruption of β-catenin binding to PTH1R affects PTH-stimulated ERK1/2 activation. We demonstrated that β-catenin interacted with wild-type PTH1R but this interaction was markedly reduced with mutant PTH1R (L584A/L585A). PTH stimulated less cAMP formation and increased more intracellular calcium in HEK293 cells transfected with wild-type PTH1R compared with mutant PTH1R, indicating β-catenin switches PTH1R signaling from Gαs activation to Gαq signaling. In addition, ERK1/2 activation in HEK293 cells transfected with PTH1R exhibited time and concentration dependence. PTH-stimulated ERK1/2 activation was mostly mediated through Gαq/PLC signaling pathway. Importantly, transfection of mutant PTH1R decreased PTH-induced ERK1/2 activation by inhibiting Gαq-mediated signaling. This study shows for the first time that the interference of β-catenin binding to PTH1R inhibits PTH-stimulated ERK1/2 phosphorylation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Scope: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis., Methods and Results: Via Oil red O staining, triglyceride, and β-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid β-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis., Conclusion: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway., (© 2015. Published by The Company of Biologists Ltd.)

Background: Fetal insulin hypothesis was proposed that the association between low birth weight and type 2 diabetes is principally genetically mediated. The aim of this study was to investigate whether common variants in genes CDKAL1, HHEX, ADCY5, SRR, PTPRD that predisposed to type 2 diabetes were also associated with reduced birthweight in Chinese Han population., Methods: Twelve single nucleotide polymorphisms (rs7756992/rs10946398 in CDKAL1, rs1111875 in HHEX, rs391300 in SRR, rs17584499 in PTPRD, rs1170806/rs9883204/rs4678017/rs9881942/rs7641344/rs6777397/rs6226243 in ADCY5) were genotyped in 1174 unrelated individuals born in Peking Union Medical College Hospital from 1921 to 1954 by TaqMan allelic discrimination assays, of which 645 had normal glucose tolerance, 181 had developed type 2 diabetes and 348 impaired glucose regulation. Associations of these 12 genetic variants with birthweight and glucose metabolism in later life were analyzed., Results: Birthweight was inversely associated with CDKAL1-rs10946398 (β = -41 g [95% confidence interval [CI]: -80, -3], P = 0.034), common variants both associated with increased risk of impaired glucose metabolism and decreased insulin secretion index later in life. After adjusting for sex, gestational weeks, parity and maternal age, the risk allele of CDKAL1-rs7756992 was associated with reduced birthweight (β = -36 g [95% CI: -72, -0.2], P = 0.048). The risk allele in SRR showed a trend toward a reduction of birthweight (P = 0.085)., Conclusions: This study identified the association between type 2 diabetes risk variants in CDKAL1 and birthweight in Chinese Han individuals, and the carrier of risk allele within SRR had the trend of reduced birthweight. This demonstrates that there is a clear overlap between the genetics of type 2 diabetes and fetal growth, which proposes that lower birth weight and type 2 diabetes may be two phenotypes of one genotype.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

Objective: To determine the contribution of ADCY5 mutations in cases with genetically undefined benign hereditary chorea (BHC)., Methods: We studied 18 unrelated cases with BHC (7 familial, 11 sporadic) who were negative for NKX2-1 mutations. The diagnosis of BHC was based on the presence of a childhood-onset movement disorder, predominantly characterized by chorea and no other major neurologic features. ADCY5 analysis was performed by whole-exome sequencing or Sanger sequencing. ADCY5 and NKX2-1 expression during brain development and in the adult human brain was assessed using microarray analysis of postmortem brain tissue., Results: The c.1252C>T; p.R418W mutation was identified in 2 cases (1 familial, 1 sporadic). The familial case inherited the mutation from the affected father, who had a much milder presentation, likely due to low-grade somatic mosaicism. The mutation was de novo in the sporadic case. The clinical presentation of these cases featured nonparoxysmal generalized chorea, as well as dystonia in the most severely affected, but no facial myokymia. We observed significant progression of symptoms in ADCY5 mutation carriers, in contrast to BHC secondary to NKX2-1 mutations. The difference in the clinical course is mirrored by the brain expression data, showing increasing ADCY5 expression in the striatum during brain development, whereas NKX2-1 shows an opposite trend., Conclusions: Our study identifies mutations in ADCY5, the gene previously linked to familial dyskinesia with facial myokymia, as a cause of familial and sporadic BHC. ADCY5 genetic analysis should be performed in cases with a benign choreiform movement disorder even in the absence of facial myokymia., (© 2015 American Academy of Neurology.)

A fraction of genetic risk to develop schizophrenia may be due to low-frequency variants. This multistep study attempted to find low-frequency variants of high effect at coding regions of eleven schizophrenia susceptibility genes supported by genome-wide association studies (GWAS) and nine genes for the DISC1 interactome, a susceptibility gene-set. During the discovery step, a total of 125 kb per sample were resequenced in 153 schizophrenia patients and 153 controls from Galicia (NW Spain), and the cumulative role of low-frequency variants at a gene or at the DISC1 gene-set were analyzed by burden and variance-based tests. Relevant results were meta-analyzed when appropriate data were available. In addition, case-only putative damaging variants were genotyped in a further 419 cases and 398 controls. The discovery step revealed a protective effect of rare missense variants at NRXN1, a result supported by meta-analysis (OR = 0.67, 95% CI: 0.47-0.94, P = 0.021, based on 3848 patients and 3896 controls from six studies). The follow-up step based on case-only putative damaging variants revealed a promising risk variant at AKAP9. This variant, K873R, reached nominal significance after inclusion of 240 additional Spanish controls from databases. The variant, located in an ADCY2 binding region, is absent from large public databases. Interestingly, GWAS revealed an association between common ADCY2 variants and bipolar disorder, a disorder with considerable genetic overlap with schizophrenia. These data suggest a role of rare missense variants at NRXN1 and AKAP9 in schizophrenia susceptibility, probably related to alteration of the excitatory/inhibitory synaptic balance, deserving further investigation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study, we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak, whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin, and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction, exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin., (Copyright © 2015 The Authors.)

The main atheroprotective mechanism of high-density lipoprotein (HDL) has been regarded as reverse cholesterol transport, whereby cholesterol from peripheral tissues is removed and transported to the liver for elimination. Although numerous additional atheroprotective mechanisms have been suggested, the role of HDL in modulating signal transduction of cell membrane-bound receptors has received little attention to date. This potential was recently highlighted following the identification of a polymorphism in the adenylyl cyclase 9 gene (ADCY9) that was shown to be a determining factor in the risk of cardiovascular (CV) events in patients treated with the HDL-raising compound dalcetrapib. Indeed, ADCY9 is part of the signaling pathway of the β2-adrenergic receptor (β2-AR) and both are membrane-bound proteins affected by changes in membrane-rich cholesterol plasma membrane domains (caveolae). Numerous G-protein-coupled receptors (GPCRs) and ion channels are affected by caveolae, with caveolae composition acting as a 'signalosome'. Polymorphisms in the genes encoding ADCY9 and β2-AR are associated with response to β2-agonist drugs in patients with asthma, malaria and with sickle cell disease. Crystallization of the β2-AR has found cholesterol tightly bound to transmembrane structures of the receptor. Cholesterol has also been shown to modulate the activity of this receptor. Apolipoprotein A1 (ApoA1), the major protein component of HDL, destabilizes and removes cholesterol from caveolae with high affinity through interaction with ATP-binding cassette transporter. Furthermore, β2-AR activity may be affected by ApoA1/HDL-targeted therapies. Taken together, these observations suggest a common pathway that potentially links a primary HDL function to the regulation of signal transduction., (Copyright © 2015 IMSS. Published by Elsevier Inc. All rights reserved.)

Cyclic diadenosine monophosphate is a recently identified signaling molecule. It has been shown to play important roles in bacterial pathogenesis. SSU98_1483 (ssDacA), which is an ortholog of Listeria monocytogenes DacA, is a putative diadenylate cyclase in Streptococcus suis serotype 2. In this study, we determined the enzymatic activity of ssDacA in vitro using high-performance liquid chromatography and mass spectrometry. Our results showed that ssDacA was a diadenylate cyclase that converts ATP into cyclic diadenosine monophosphate in vitro. The diadenylate cyclase activity of ssDacA was dependent on divalent metal ions such as Mg(2+), Mn(2+), or Co(2+), and it is more active under basic pH than under acidic pH. The conserved RHR motif in ssDacA was essential for its enzymatic activity, and mutation in this motif abolished the diadenylate cyclase activity of ssDacA. These results indicate that ssDacA is a diadenylate cyclase, which synthesizes cyclic diadenosine monophosphate in Streptococcus suis serotype 2.

Cyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Aim: Pharmacogenetic studies continue to search for pretreatment predictors of chemotherapeutic efficacy and toxicity in metastatic colorectal cancer. Both genome-wide association studies and candidate gene studies have yielded potential genetic markers for chemosensitivity. We conducted a clinical association study, validating the effect of specific genetic markers cited in recently published papers on the efficacy of the oral 5-fluoro-uracil prodrug capecitabine., Patients and Methods: Germline DNA was collected for 268 metastatic colorectal cancer patients from the CAIRO trial, a multicenter phase III trial, randomizing between combined or sequential first-line treatment with capecitabine, irinotecan, and oxaliplatin. Genotyping was performed for eight single-nucleotide polymorphisms (SNPs), using high-resolution melting curves. Four SNPs are located in the MTRR gene, and another four SNPs showed significant association with 5-fluoro-uracil cytotoxicity in a recent in-vitro genome-wide association study. The primary endpoint was progression-free survival (PFS); secondary endpoints were objective response and overall survival., Results: In patients receiving capecitabine monotherapy, rs4702484, located in ADCY2 and close to MTRR, was associated with slightly reduced PFS for homozygous wild-type patients (CC 6.2 vs. CT 8.0 months; P=0.018). For the other selected genetic markers, we found no association with PFS, overall survival, or radiologic response upon treatment with capecitabine, either in the total study population or in the capecitabine monotherapy subgroup., Conclusion: With the exception of rs4702484, we found no evidence of an effect on capecitabine chemosensitivity for any of the studied SNPs. More specifically, variants in methionine synthase reductase (MTRR) are not likely associated with capecitabine efficacy.

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation. Males and females, age groups 20-30 years and 50-70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6h, p=0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p=0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases., (Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Alterations in vascular smooth muscle cells (SMCs) contribute to the pathogenesis of intracranial aneurysms (IAs), but the genetic mechanisms underlying these alterations are unclear. We used microarray analysis to compare tissue small noncoding RNA and messenger RNA expression profiles in vessel wall samples from patients with late-stage IAs. We identified myocardin (MYOCD), a key contractility regulator of vascular SMCs, as a critical factor in IA progression. Using a multifaceted computational and experimental approach, we determined that depletion of competitive endogenous RNAs (ARHGEF12, FGF12, and ADCY5) enhanced factors that downregulate MYOCD, which induces the conversion of SMCs from differentiated contractile states into dedifferentiated phenotypes that exhibit enhanced proliferation, synthesis of new extracellular matrix, and organization of mural thrombi. These effects may lead to the repair and maintenance of IAs. This study presents guidelines for the prediction and validation of the IA regulator MYOCD in competitive endogenous RNA networks and facilitates the development of novel therapeutic and diagnostic tools for IAs.

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

The evolution of multicellularity required novel mechanisms for intercellular communication, but their origin is unclear. Dictyostelium cells exchange signals to position specialized cell types in multicellular spore-bearing structures. These signals activate complex pathways that converge on activation of cAMP-dependent protein kinase (PKA). Genes controlling PKA were detected in the Dictyostelid unicellular ancestors, which like most protists form dormant cysts when experiencing environmental stress. We deleted PKA and the adenylate cyclases AcrA and AcgA, which synthesize cAMP for PKA activation, in the intermediate species Polysphondylium, which can develop into either cysts or into multicellular structures. Loss of PKA prevented multicellular development, but also completely blocked encystation. Loss of AcrA and AcgA, both essential for sporulation in Dictyostelium, did not affect Polysphondylium sporulation, but prevented encystation. We conclude that multicellular cAMP signalling was co-opted from PKA regulation of protist encystation with progressive refunctionalization of pathway components.

The Yesso scallop Patinopecten yessoensis is an economically important marine bivalve species in aquaculture and fishery in Asian countries. However, limited genomic resources are available for this scallop, which hampers investigations into molecular mechanisms underlying their unique biological characteristics, such as shell formation and pigmentation. Mantle is the special tissue of P. yessoensis that secretes biomineralization proteins inducing shell deposition as well as pigmentation on the shells. However, a current deficiency of transcriptome information limits insight into mechanisms of shell formation and pigmentation in this species. In this study, the transcriptome of the mantle of P. yessoensis was deeply sequenced and characterized using Illumina RNA-seq technology. A total of 86,521 unique transcripts are assembled from 55,884,122 reads that passed quality filters, and annotated, using Gene Ontology classification. A total of 259 pathways are identified in the mantle transcriptome, including the calcium signaling and melanogenesis pathways. A total of 237 unigenes that are homologous to 102 reported biomineralization genes are identified, and 121 unigenes that are homologous to 93 known proteins related to melanin biosynthesis are found. Twenty-three annotated unigenes, which are mainly homologous to calmodulin and related proteins, Ca2+/calmodulin-dependent protein kinase, adenylate/guanylate cyclase, and tyrosinase family are potentially involved in both biomineralization and melanin biosynthesis. It is suggested that these genes are probably not limited in function to induce shell deposition by calcium metabolism, but may also be involved in pigmentation of the shells of the scallop. This potentially supports the idea that there might be a link between calcium metabolism and melanin biosynthesis, which was previously found in vertebrates. The findings presented here will notably advance the understanding of the sophisticated processes of shell formation as well as shell pigmentation in P. yessoensis and other bivalve species, and also provide new evidence on gene expression for the understanding of pigmentation and biomineralization not only in invertebrates but also probably in vertebrates.

Cyclic AMP signaling is critical for activity-dependent refinement of neuronal circuits. Global disruption of adenylyl cyclase 1 (AC1), the major calcium/calmodulin-stimulated adenylyl cyclase in the brain, impairs formation of whisker-related discrete neural modules (the barrels) in cortical layer 4 in mice. Since AC1 is expressed both in the thalamus and the neocortex, the question of whether pre- or postsynaptic (or both) AC1 plays a role in barrel formation has emerged. Previously, we generated cortex-specific AC1 knockout (Cx-AC1KO) mice and found that these animals develop histologically normal barrels, suggesting a potentially more prominent role for thalamic AC1 in barrel formation. To determine this, we generated three new lines of mice: one in which AC1 is disrupted in nearly half of the thalamic ventrobasal nucleus cells in addition to the cortical excitatory neurons (Cx/pTh-AC1KO mouse), and another in which AC1 is disrupted in the thalamus but not in the cortex or brainstem nuclei of the somatosensory system (Th-AC1KO mouse). Cx/pTh-AC1KO mice show severe deficits in barrel formation. Th-AC1KO mice show even more severe disruption in barrel patterning. In these two lines, single thalamocortical (TC) axon labeling revealed a larger lateral extent of TC axons in layer 4 compared to controls. In the third line, all calcium-stimulated adenylyl cyclases (both AC1 and AC8) are deleted in cortical excitatory neurons. These mice have normal barrels. Taken together, these results indicate that thalamic AC1 plays a major role in patterning and refinement of the mouse TC circuitry., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aims/hypothesis: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an important role in glucose-induced signalling and glucose homeostasis., Methods: We used pharmacological and genetic approaches in beta cells to determine secretion and calcium metabolism. Furthermore, Adcy8 knockout mice were characterised., Results: In clonal beta cells, inhibitors of adenylyl cyclases or their downstream targets reduced the glucose-induced increase in cytosolic calcium and insulin secretion. This was reproduced by knock-down of ADCY8, but not of ADCY1. These agents also inhibited glucose-induced increase in cytosolic calcium and electrical activity in primary beta cells and similar effects were observed after ADCY8 knock-down. Moreover, insulin secretion was diminished in islets from Adcy8 knockout mice. These mice were glucose intolerant after oral or intraperitoneal administration of glucose whereas their levels of glucagon-like peptide-1 remained unaltered. Finally, we knocked down ADCY8 in the ventromedial hypothalamus to evaluate the need for ADCY8 in the central regulation of glucose homeostasis. Whereas mice fed a standard diet had normal glucose levels, high-fat diet exacerbated glucose intolerance and knock-down mice were incapable of raising their plasma insulin levels. Finally we confirmed that ADCY8 is expressed in human islets., Conclusions/interpretations: Collectively, our findings demonstrate that ADCY8 is required for the physiological activation of glucose-induced signalling pathways in beta cells, for glucose tolerance and for hypothalamic adaptation to a high-fat diet via regulation of islet insulin secretion.

Background: Dalcetrapib did not improve clinical outcomes, despite increasing high-density lipoprotein cholesterol by 30%. These results differ from other evidence supporting high-density lipoprotein as a therapeutic target. Responses to dalcetrapib may vary according to patients' genetic profile., Methods and Results: We conducted a pharmacogenomic evaluation using a genome-wide approach in the dal-OUTCOMES study (discovery cohort, n=5749) and a targeted genotyping panel in the dal-PLAQUE-2 imaging trial (support cohort, n=386). The primary endpoint for the discovery cohort was a composite of cardiovascular events. The change from baseline in carotid intima-media thickness on ultrasonography at 6 and 12 months was evaluated as supporting evidence. A single-nucleotide polymorphism was found to be associated with cardiovascular events in the dalcetrapib arm, identifying the ADCY9 gene on chromosome 16 (rs1967309; P=2.41×10(-8)), with 8 polymorphisms providing P<10(-6) in this gene. Considering patients with genotype AA at rs1967309, there was a 39% reduction in the composite cardiovascular endpoint with dalcetrapib compared with placebo (hazard ratio, 0.61; 95% confidence interval, 0.41-0.92). In patients with genotype GG, there was a 27% increase in events with dalcetrapib versus placebo. Ten single-nucleotide polymorphism in the ADCY9 gene, the majority in linkage disequilibrium with rs1967309, were associated with the effect of dalcetrapib on intima-media thickness (P<0.05). Marker rs2238448 in ADCY9, in linkage disequilibrium with rs1967309 (r(2)=0.8), was associated with both the effects of dalcetrapib on intima-media thickness in dal-PLAQUE-2 (P=0.009) and events in dal-OUTCOMES (P=8.88×10(-8); hazard ratio, 0.67; 95% confidence interval, 0.58-0.78)., Conclusions: The effects of dalcetrapib on atherosclerotic outcomes are determined by correlated polymorphisms in the ADCY9 gene., Clinical Trial Information: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00658515 and NCT01059682., (© 2015 American Heart Association, Inc.)

Background: Several studies have investigated the effect of known adult body mass index (BMI) associated single nucleotide polymorphisms (SNPs) on BMI in childhood. There has been no genome-wide association study (GWAS) of BMI trajectories over childhood., Methods: We conducted a GWAS meta-analysis of BMI trajectories from 1 to 17 years of age in 9377 children (77,967 measurements) from the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Western Australian Pregnancy Cohort (Raine) Study. Genome-wide significant loci were examined in a further 3918 individuals (48,530 measurements) from Northern Finland. Linear mixed effects models with smoothing splines were used in each cohort for longitudinal modelling of BMI., Results: A novel SNP, downstream from the FAM120AOS gene on chromosome 9, was detected in the meta-analysis of ALSPAC and Raine. This association was driven by a difference in BMI at 8 years (T allele of rs944990 increased BMI; PSNP = 1.52 × 10(-8)), with a modest association with change in BMI over time (PWald(Change) = 0.006). Three known adult BMI-associated loci (FTO, MC4R and ADCY3) and one childhood obesity locus (OLFM4) reached genome-wide significance (PWald < 1.13 × 10(-8)) with BMI at 8 years and/or change over time., Conclusions: This GWAS of BMI trajectories over childhood identified a novel locus that warrants further investigation. We also observed genome-wide significance with previously established obesity loci, making the novel observation that these loci affected both the level and the rate of change in BMI. We have demonstrated that the use of repeated measures data can increase power to allow detection of genetic loci with smaller sample sizes., (© The Author 2015; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)