TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Members of the KCTD family are major regulators of cAMP signaling.

Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger with an essential role in neuronal function. cAMP synthesis by adenylyl cyclases (AC) is controlled by G protein-coupled receptor (GPCR) signaling systems. However, the network of molecular players involved in the process is incompletely defined. Here, we used CRISPR/Cas9-based screening to identify that members of the potassium channel tetradimerization domain (KCTD) family are major regulators of cAMP signaling. Focusing on striatal neurons, we show that the dominant isoform KCTD5 exerts its effects through an unusual mechanism that modulates the influx of Zn2+ via the Zip14 transporter to exert unique allosteric effects on AC. We further show that KCTD5 controls the amplitude and sensitivity of stimulatory GPCR inputs to cAMP production by Gßγ-mediated AC regulation. Finally, we report that KCTD5 haploinsufficiency in mice leads to motor deficits that can be reversed by chelating Zn2+ Together, our findings uncover KCTD proteins as major regulators of neuronal cAMP signaling via diverse mechanisms.

An oral GLP-1 and GIP dual receptor agonist improves metabolic disorders in high fat-fed mice.

Dual activation of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor has potential as a novel strategy for treatment of diabesity. Here, we created a hybrid peptide which we named 19W, and show that it is more stable in presence of murine plasma than exendin-4 is. In vitro studies were performed to reveal that 19W could stimulate insulin secretion from INS-1 cells in a dose-dependent manner, just like the native peptide GIP and exendin-4 do. 19W effectively evoked dose-dependent cAMP production in cells targeting both GLP-1R and GIPR. In healthy C57BL/6J mice, the single administration of 19W significantly improved glucose tolerance. When administered in combination with sodium deoxycholate (SDC), its oral hypoglycemic activity was enhanced. Pharmacokinetics studies in Wistar rats revealed that 19W was absorbed following oral uptake, while SDC increased its bioavailability. A long-term (28 days) exposure study of twice-daily oral administration to high fat-fed (HFF) mice showed that 19W significantly reduced animal food intake, body weight, fasting blood glucose, total serum cholesterol (T-CHO), non-esterified free fatty acids (NEFA), and low-density lipoprotein cholesterol (LDL-C) levels. It also significantly improved glucose tolerance and the pancreatic ß/α cell ratio, and decreased the area of liver fibrosis. These results clearly demonstrate the beneficial action of this novel oral GLP-1/GIP dual receptor agonist to reduce adiposity and hyperglycemia in diabetic mice and to ameliorate liver fibrosis associated with obesity. This dual-acting peptide can be considered a good candidate for novel oral therapy to treat obesity and diabetes.

Liquiritigenin enhances cyclic adenosine monophosphate production to mitigate inflammation in dendritic cells.

OBJECTIVE: This study aims to dissect the mechanism of traditional Chinese medicinal herbs against asthma; we chose to first focus on the main chemical components of licorice to investigate their contribution to asthmatic inflammation inhibition. METHODS: Production of cellular nucleotide molecules such as cAMP, cGMP, and cGAMP was examined by using enzyme-linked immunosorbent assay (ELISA). Enzyme-encoding genes were tested in vitro using quantitative real-time PCR and protein level was detected by Western blotting analysis. In addition, co-culturing of murine dendritic cells together with T cells was conducted to examine the expression of cytokine genes and host immune response. RESULTS: We found that one of the components within licorice, named liquiritigenin (LR), could efficiently enhance cAMP production in different cell lines. The augmentation of such molecules was linked to the high expression of cAMP synthesis genes and repressed expression of cAMP breaking down genes. In addition, the downstream immune response was also alleviated by the increase in cAMP levels by LR, suggesting the great potential of this molecule against inflammation. Subsequent immunological tests showed that LR could efficiently inhibit the expression of several cytokines and alter the NF-κB pathway and T cell polarization. CONCLUSION: Altogether, we have identified a promising antiasthmatic agent LR that could exhibit immunosuppressive function by elevating the cAMP level.

Enhanced ATP and antioxidant levels for cAMP biosynthesis by Arthrobacter sp. CCTCC 2013431 with polyphosphate addition.

OBJECTIVES: When citrate and pyruvate were utilized to strengthen ATP generation for high cAMP production, oxidative stress became more severe in cells resulting in lower cell viability and cAMP formation at the late fermentation phase. To further improve cAMP biosynthesis, the effects of polyphosphate on cAMP fermentation performance together with intracellular ATP and oxidation levels were investigated under high oxidative stress condition and then high efficient cAMP fermentation process based on polyphosphate and salvage synthesis was developed and studied. RESULTS: With 2 g/L-broth sodium hexametaphosphate added at 24 h was determined as the optimal condition for cAMP production by Arthrobacter sp. CCTCC 2013431 in shake flasks. Under high oxidative stress condition caused by adding 15 mg/L-broth menadione, cAMP contents and cell viability were improved greatly due to hexametaphosphate addition and also exceeded those of control (without hexametaphosphate and menadione added) when fermentations were conducted in a 7 L bioreactor. Meanwhile, ATP levels and antioxidant capacity were improved obviously by hexametaphosphate as well. Moreover, a fermentation process with hexametaphosphate and hypoxanthine coupling added was developed by which cAMP concentration reached 7.25 g/L with an increment of 87.1% when compared with only hypoxanthine added batch and the high ROS contents generated from salvage synthesis were reduced significantly. CONCLUSION: Polyphosphate could improve intracellular ATP levels and antioxidant capacity significantly under high oxidative stress condition resulting in enhanced cell viability and cAMP fermentation production no matter by de novo synthesis or salvage synthesis.

ß-Agonist exposure preferentially impacts lung macrophage cyclic AMP-related gene expression in asthma and asthma COPD overlap syndrome.

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged ß-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute ß-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression (P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

GLP-1 reduces the migration of hepatocellular carcinoma cells via suppression of the stress-activated protein kinase/c-Jun N-terminal kinase pathway.

Incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones secreted from small intestine accompanied with oral intake. We previously showed that transforming growth factor (TGF)-α stimulates the migration of hepatocellular carcinoma (HCC) cells via mitogen-activated protein (MAP) kinases, AKT and Rho-kinase. However, it remains to be elucidated whether incretins affect HCC cell functions. In the present study, therefore, we investigated whether incretins affect the migration of HCC cells using human HCC-derived HuH7 cells. GLP-1, but not GIP, reduced both TGF-α- and hepatocyte growth factor (HGF)-induced cell migration. IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, enhanced the suppressive effect of GLP-1. GLP-1 attenuated the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by TGF-α and HGF. Our results strongly suggest that GLP-1 suppresses TGF-α- and HGF-induced migration of HCC cells through inhibiting the SAPK/JNK signaling pathway, and that the inhibition by GLP-1 is due to cAMP production.

Elucidation of the dimeric interplay of dual MRAP2 proteins in the zebrafish.

The melanocortin receptor accessory protein 2 (MRAP2) plays an essential role in the regulation of metabolic homeostasis and deletion of which results in severe obesity syndrome in mice and human. Mammalian MRAP2 is recognized as an endogenous physiological mediator through the potentiation of the MC4R signaling in vivo. Two isoforms of MRAP2 are identified in zebrafish genome, zMRAP2a and zMRAP2b. However, the mechanism of assembling dual topology and the regulatory roles of each complex on the melanocortin cascades remains unclear. In this study, we showed the bidirectional homo- and hetero-dimeric topologies of two zebrafish MRAP2 isoforms on the plasma membrane. Orientation fixed chimeric proteins could affect the trafficking and pharmacological properties of zMC4R signaling. Reciprocal replacement of zMRAP2a and zMRAP2b proteins elucidated the major participation of the carboxyl terminal as the functional domain for modulating zMC4R signaling. Our findings revealed the complex and dynamic conformational regulation of dual zebrafish MRAP2 proteins in vitro.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats.

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

A metabolically stable apelin-17 analog decreases AVP-induced antidiuresis and improves hyponatremia.

Apelin and arginine-vasopressin (AVP) are conversely regulated by osmotic stimuli. We therefore hypothesized that activating the apelin receptor (apelin-R) with LIT01-196, a metabolically stable apelin-17 analog, may be beneficial for treating the Syndrome of Inappropriate Antidiuresis, in which AVP hypersecretion leads to hyponatremia. We show that LIT01-196, which behaves as a potent full agonist for the apelin-R, has an in vivo half-life of 156 minutes in the bloodstream after subcutaneous administration in control rats. In collecting ducts, LIT01-196 decreases dDAVP-induced cAMP production and apical cell surface expression of phosphorylated aquaporin 2 via AVP type 2 receptors, leading to an increase in aqueous diuresis. In a rat experimental model of AVP-induced hyponatremia, LIT01-196 subcutaneously administered blocks the antidiuretic effect of AVP and the AVP-induced increase in urinary osmolality and induces a progressive improvement of hyponatremia. Our data suggest that apelin-R activation constitutes an original approach for hyponatremia treatment.

Inhibitory effect of UDP-glucose on cAMP generation and insulin secretion.

Type-2 diabetes (T2D) is a global disease caused by the inability of pancreatic ß-cells to secrete adequate insulin. However, the molecular mechanisms underlying the failure of ß-cells to respond to glucose in T2D remains unknown. Here, we investigated the relative contribution of UDP-glucose (UDP-G), a P2Y14-specific agonist, in the regulation of insulin release using human isolated pancreatic islets and INS-1 cells. P2Y14 was expressed in both human and rodent pancreatic ß-cells. Dose-dependent activation of P2Y14 by UDP-G suppressed glucose-stimulated insulin secretion (GSIS) and knockdown of P2Y14 abolished the UDP-G effect. 12-h pretreatment of human islets with pertussis-toxin (PTX) improved GSIS and prevented the inhibitory effect of UDP-G on GSIS. UDP-G on GSIS suppression was associated with suppression of cAMP in INS-1 cells. UDP-G decreased the reductive capacity of nondiabetic human islets cultured at 5 mm glucose for 72 h and exacerbated the negative effect of 20 mm glucose on the cell viability during culture period. T2D donor islets displayed a lower reductive capacity when cultured at 5 mm glucose for 72 h that was further decreased in the presence of 20 mm glucose and UDP-G. Presence of a nonmetabolizable cAMP analog during culture period counteracted the effect of glucose and UDP-G. Islet cultures at 20 mm glucose increased apoptosis, which was further amplified when UDP-G was present. UDP-G modulated glucose-induced proliferation of INS-1 cells. The data provide intriguing evidence for P2Y14 and UDP-G's role in the regulation of pancreatic ß-cell function.

Discovery of substituted 3H-pyrido[2,3-d]pyrimidin-4-ones as potent, biased, and orally bioavailable sst2 agonist.

The discovery of a novel 3H-pyrido[2,3-d]pyrimidin-4-one series as potent and biased sst2 agonists is described. This class of molecules exhibits excellent sst2 potency and selectivity against sst1, sst3, and sst5 receptors, and they are significantly more potent at inhibiting cAMP production than inducing internalization. The orally bioavailable 6-(3-chloro-5-methylphenyl)-3-(3-fluoro-5-hydroxyphenyl)-5-({methyl[(2S)-pyrrolidin-2-ylmethyl]amino}methyl)-3H,4H-pyrido[2,3-d]pyrimidin-4-one (36) also suppresses GH secretion in GHRH-challenged rats in a dose-dependent manner.

Functional characterization of a C-terminal splice variant of the human melanocortin 1 receptor.

The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and sensitivity to ultraviolet radiation. When stimulated by its natural agonists, it promotes the switch from synthesis of poorly photoprotective and lightly colored pheomelanins to production of photoprotective and darker eumelanins. In addition to an unusually high number of single nucleotide polymorphisms, the MC1R is expressed as 3 protein-coding splice variants. Two transcripts display different 5' untranslated sequences but yield the same open reading frame corresponding to the canonical 317 aminoacids protein (termed MC1R). An alternative transcript named MC1R-203 encodes for a 382 amino acids protein of poorly characterized functional properties containing an additional 65 aminoacids C-terminal extension. Given the known roles of the MC1R C-terminal extension in forward trafficking, coupling to intracellular effectors and desensitization, the different structure of this domain in MC1R and MC1R-203 may lead to significant functional alteration(s). We have assessed the functional properties of MC1R-203, as compared with the canonical MC1R form. We show that unstimulated HBL human melanoma cells express the MC1R-203 spliceoform, although at much lower levels than canonical MC1R. When expressed in heterologous HEK293 cells, the presence of the 65 aminoacid-long cytosolic extension immediately after Cys316 in MC1R-203 did not impair the intracellular stability of the protein, but it interfered with functional coupling to the cAMP cascade and with the ubiquitylation of ARRB2 associated with MC1R desensitization. Conversely, MC1R-203 retained full capacity to activate ERK1/2 signaling. Accordingly, MC1R-203 displays biased signaling when expressed in HEK293 cells.

Allosteric Antagonism of the A2A Adenosine Receptor by a Series of Bitopic Ligands.

Allosteric antagonism by bitopic ligands, as reported for many receptors, is a distinct modulatory mechanism. Although several bitopic A2A adenosine receptor (A2AAR) ligand classes were reported as pharmacological tools, their receptor binding and functional antagonism patterns, i.e., allosteric or competitive, were not well characterized. Therefore, here we systematically characterized A2AAR binding and functional antagonism of two distinct antagonist chemical classes. i.e., fluorescent conjugates of xanthine amine congener (XAC) and SCH442416. Bitopic ligands were potent, weak, competitive or allosteric, based on the combination of pharmacophore, linker and fluorophore. Among antagonists tested, XAC, XAC245, XAC488, SCH442416, MRS7352 showed Ki binding values consistent with KB values from functional antagonism. Interestingly, MRS7396, XAC-X-BY630 (XAC630) and 5-(N,N-hexamethylene)amiloride (HMA) were 9-100 times weaker in displacing fluorescent MRS7416 binding than radioligand binding. XAC245, XAC630, MRS7396, MRS7416 and MRS7322 behaved as allosteric A2AAR antagonists, whereas XAC488 and MRS7395 antagonized competitively. Schild analysis showed antagonism slopes of 0.42 and 0.47 for MRS7396 and XAC630, respectively. Allosteric antagonists HMA and MRS7396 were more potent in displacing [3H]ZM241385 binding than MRS7416 binding. Sodium site D52N mutation increased and decreased affinity of HMA and MRS7396, respectively, suggesting possible preference for different A2AAR conformations. The allosteric binding properties of some bitopic ligands were rationalized and analyzed using the Hall two-state allosteric model. Thus, fluorophore tethering to an orthosteric ligand is not neutral pharmacologically and may confer unexpected properties to the conjugate.

Membrane-Associated α-Tubulin Is Less Acetylated in Postmortem Prefrontal Cortex from Depressed Subjects Relative to Controls: Cytoskeletal Dynamics, HDAC6, and Depression.

Cytoskeletal proteins and post-translational modifications play a role in mood disorders. Post-translational modifications of tubulin also alter microtubule dynamics. Furthermore, tubulin interacts closely with Gαs, the G-protein responsible for activation of adenylyl cyclase. Postmortem tissue derived from depressed suicide brain showed increased Gαs in lipid-raft domains compared with normal subjects. Gαs, when ensconced in lipid rafts, couples less effectively with adenylyl cyclase to produce cAMP, and this is reversed by antidepressant treatment. A recent in vitro study demonstrated that tubulin anchors Gαs to lipid rafts and that increased tubulin acetylation (due to HDAC6 inhibition) and antidepressant treatment decreased the proportion of Gαs complexed with tubulin. This suggested that deacetylated-tubulin might be more prevalent in depression. This study examined tubulin acetylation in whole-tissue homogenate, plasma membrane, and lipid-raft membrane domains in tissue from normal control subjects, depressed suicides, and depressed nonsuicides (human males/females). While tissue homogenate showed no changes in tubulin acetylation between control, depressed suicides, and depressed nonsuicides, plasma membrane-associated tubulin showed significant decreases in acetylation from depressed suicides and depressed nonsuicides compared with controls. No change was seen in expression of the enzymes responsible for tubulin acetylation or deacetylation. These data suggest that, during depression, membrane-localized tubulin maintains a lower acetylation state, permitting increased sequestration of Gαs in lipid-raft domains, where it is less likely to couple to adenylyl cyclase for cAMP production. Thus, membrane tubulin may play a role in mood disorders, which could be exploited for diagnosis and treatment.SIGNIFICANCE STATEMENT There is little understanding about the molecular mechanisms involved in the development of depression and, in severe cases, suicide. Evidence for the role of microtubule modifications in progression of depressive disorders is emerging. These postmortem data provide strong evidence for membrane tubulin modification leading to reduced efficacy of the G protein, Gαs, in depression. This study reveals a direct link between decreased tubulin acetylation in human depression and the increased localization of Gαs in lipid-raft domains responsible for attenuated cAMP signaling. The evidence presented here suggest a novel diagnostic and therapeutic locus for depression.

Agonist Effects of Propranolol on Non-Tumor Human Breast Cells.

The ß-blocker propranolol (PROP) has been proposed as a repurposed treatment for breast cancer. The similarity of action between ß-agonists and antagonists found on breast cells encouraged us to compare PROP and isoproterenol (ISO, agonist) signaling pathways on a human breast cell line. Cell proliferation was measured by cell counting and DNA-synthesis. Cell adhesion was measured counting the cells that remained adhered to the plastic after different treatments. Changes in actin cytoskeleton were observed by fluorescence staining and Western Blot. ISO and PROP caused a diminution of cell proliferation and an increase of cell adhesion, reverted by the pure ß-antagonist ICI-118551. ISO and PROP induced a reorganization of actin cytoskeleton increasing F-actin, p-COFILIN and p-LIMK. While ISO elicited a marked enhancement of cAMP concentrations and an increase of vasodilator-stimulated phosphoprotein (VASP) and cAMP response element-binding protein (CREB) phosphorylation, PROP did not. Clathrin-mediated endocytosis inhibition or ß-arrestin1 dominant-negative mutant abrogated PROP-induced cell adhesion and COFILIN phosphorylation. The fact that PROP has been proposed as an adjuvant drug for breast cancer makes it necessary to determine the specific action of PROP in breast models. These results provide an explanation for the discrepancies observed between experimental results and clinical evidence.

An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.

Topical treatment strategies to manipulate human skin pigmentation.

Skin pigmentation is a result of melanin produced by melanocytes in the epidermis. Melanocyte activity, along with the type and distribution of melanins, is the main driver for diversity of skin pigmentation. Dark melanin acts to protect against the deleterious effects of ultraviolet (UV) radiation, including photo-aging and skin cancer formation. In turn, UV radiation activates skin melanocytes to induce further pigmentation (i.e., "tanning pathway"). The well-characterized MSH/MC1R-cAMP-MITF pathway regulates UV-induced melanization. Pharmacologic activation of this pathway ("sunless tanning") represents a potential strategy for skin cancer prevention, particularly in those with light skin or the "red hair" phenotype who tan poorly after UV exposure due to MC1R inactivating polymorphisms. Skin hyperpigmentation can also occur as a result of inflammatory processes and dermatological disorders such as melasma. While primarily of cosmetic concern, these conditions can dramatically impact quality of life of affected patients. Several topical agents are utilized to treat skin pigmentation disorders. Here, we review melanogenesis induced by UV exposure and the agents that target this pathway.

In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the ß-arrestin2 recruitment assay: No two ligands have the same profile.

Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a ß-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(µ), DOP(δ) or KOP(κ) receptors(-r). Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist; its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r; its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP. For the ß-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit ß-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for ß-arrestin2 recruitment to the DOP-r and KOP-r respectively. Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting ß-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for ß-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production.

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.