Phosphodiesterase Enzyme Research Articles

Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

Comparison of Effect of Leukotriene Biosynthesis Blockers and Inhibitors of Phosphodiesterase Enzyme in Patients with Bronchial Hyperreactivity.

AIM:Blocking effect of leukotriene biosynthesis–zileuton and blocking the effect of phosphodiesterase enzyme–diprophylline in the treatment of patients with bronchial asthma and bronchial increased reactivity, and tiotropium bromide as an antagonist of the muscarinic receptor studied in this work.METHODS:Parameters of the lung function are determined with Body plethysmography. The resistance of the airways (Raw) was registered and measured was intrathoracic gas volume (ITGV), and specific resistance (SRaw) was also calculated. For the research, administered was zileuton (tabl. 600 mg) and diprophylline (tabl. 150 mg).RESULTS:Two days after in-house administration of leukotriene biosynthesis blocker–zileuton (4 x 600 mg orally), on the day 3 initial values of patients measured and afterwards administered 1 tablet of zileuton, and again measured was Raw and ITGV, after 60, 90 and 120 min. and calculated was SRaw; (p < 0.01). Diprophylline administered 7 days at home in a dose of (2 x 150 mg orally), on the day 8 to same patients administered 1 tablet of diprophylline, and performed measurements of Raw, ITGV, after 60, 90 and 120 min, and calculated the SRaw (p < 0.05). Treatment of the control group with tiotropium bromide - antagonist of the muscarinic receptor (2 inh. x 0.18 mcg), is effective in removal of the increased bronchomotor tonus, by also causing the significant decrease of the resistance (Raw), respectively of the specific resistance (SRaw), (p < 0.05).CONCLUSION:Effect of zileuton in blocking of leukotriene biosynthesis is not immediate after oral administration, but the effect seen on the third day of cys-LTs’ inhibition, and leukotriene B4 (LTB4) and A4 (LTA4) in patients with bronchial reactivity and bronchial asthma, which is expressed with a high significance, (p < 0.01). Blockage of phosphodiesterase enzyme–diprophylline decreases the bronchial reactivity, which is expressed with a moderate significance, (p < 0.05).

Effects of α-(prazosin and yohimbine) and β-receptors activity on cAMP generation and UCP1 gene expression in brown adipocytes.

Abstract Background Brown adipose tissue (BAT) contains both α- and β-adrenergic receptors. In the literature, the activity of α-adrenoreceptors is less documented, and their functions still remain puzzling. The present investigation has been undertaken to understand α-adrenoreceptors’ activity and their relation between uncoupling protein 1 (UCP1) mRNA expression and cyclic AMP (cAMP) generation in BAT. Methods BAT precursor cells from young mice were grown in culture. Cells were exposed to norepinephrine (NE) and other agents. RNA was isolated after harvesting the cells, and northern blot was performed. Filters were exposed to film after hybridization with nick-translated complementary DNA probes, and results were evaluated by scanning. Amersham assay kit was used for cAMP measurement. Results Treatment of prazosin and yohimbine separately with 1 μM of NE shows stimulation of UCP1 mRNA expression 106% and 154%, respectively, whereas with that of both drugs shows only 76%. cAMP generation occurs 282% with prazosin, 100% with yohimbine, and 382% with both drugs with 1 μM of NE, whereas it is 310%, 40%, and 358%, respectively, with 10 μM of NE. Conclusions Stimulation of thermogenesis after treatment of prazosin and NE may be due to the inhibition of phosphodiesterase enzyme and with yohimbine and NE indicates the possibility of inhibition of the inhibitory effect of α2- and stimulation of α1-receptors. Increase of cAMP concentration with yohimbine and both drugs with NE are not correlated to UCP1 mRNA expression. This indicates that the relationship between cAMP elevation and stimulation of thermogenesis is not simple. This study clearly shows the interaction between β- and α-adrenoreceptor activities.

Soluble Epoxide Hydrolase/Phosphodiesterase Dual Inhibitor is Efficacious Against Inflammatory Pain

The soluble epoxide hydrolase (sEH) is the principal regulator of epoxy fatty‐acids (EpFA) transforming them in to the less active diols. Inhibitors of sEH block this activity and maintain levels of EpFA in vivo which has demonstrated potent analgesia in several preclinical models of acute and chronic pain. However, sEH inhibitors have no observable action in the absence of a pain state [1]. It was therefore hypothesized that an additional factor was present in the painful state that was required for the antinociceptive action of the EpFA [2]. Investigation began with cyclic adenosine monophosphate (cAMP) and experiments demonstrated this was active but also that increasing cAMP with phosphodiesterase inhibitors increased EpFA as well. Phosphodiesterase enzymes hydrolyze the cyclic nucleotide into adenosine monophosphate altering its secondary messenger capacity. This led to the design and synthesis of several dual inhibitors of sEH and phosphodiesterase.Multi‐target ligands are designed to improve safety and efficacy in treatment of diseases. The use of drug combinations cannot be assumed to be safe given drug‐drug interactions. Additionally, the prediction of pharmacodynamic and pharmacokinetic relationships is less complicated with a single entity. The multi‐target ligands are selected to improve therapeutic efficacy by hitting multiple targets in physiological disorders. Thus, increasing the EpFA by halting their degradation and at the same time potentiating their activity with an increase in cAMP was the goal with synthesis of the dual inhibitor. We present the biochemical characterization as well as the in vivo efficacy in an inflammatory pain model for the selected dual inhibitor of sEH and PDE enzymes. The IC50 of RBH61 for sEH determined on the sEH human recombinant enzyme is 2.1 ± 0.5 nM. This is paired with a IC50 in the PDE assay of 8.1 ± 0.05 nM in human embryonic kidney (HEK293) cells. Evaluation of the dual inhibitor in a model of inflammatory pain in rats demonstrates its antinociceptive efficacy.Support or Funding InformationThis work was supported, in part, by National Institutes of Health grants: NIEHS R01 ES002710 and NIEHS/Superfund Research Program P42 ES004699, NINDS U54 NS079202 and Grants NIEHS T32ES007059, NIH 5T32DC008072‐05 and 4T32HL086350‐09.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S43. A PROOF-OF-MECHANISM STUDY OF THE PDE10 INHIBITOR RG7203 IN PATIENTS WITH PROBING REWARD FUNCTIONS WITH IMAGING AND BEHAVIORAL APPROACHES

BackgroundThe enzyme phosphodiesterase 10A (PDE10A) is highly expressed in the striatum where it modulates both dopamine D2 and D1 dependent signaling. Its inhibition leads to a suppression of D2 mediated signaling –similar to effects of D2 antagonists - and an enhancement of D1 dependent signaling. D1-dependent signaling has been implicated in reward based learning. Its deficient activation may be a key factor underlying deficient reward functions including reward anticipation and reward based learning that have been implicated as major drivers of negative symptoms of schizophrenia. Therefore, inhibition of PDE10 could be a way to ameliorate such deficits and consequently negative symptoms. In healthy volunteers the PDE10 inhibitor RG7203 indeed enhanced performance in tasks that probed reward functioning suggestive of its potential utility to treat negative symptoms in schizophrenia. We therefore tested the hypothesis that it should enhance imaging and behavioral markers of reward functions in patients with moderate negative symptoms in order to establish mechanistic proof of its utility as treatment of negative symptoms.MethodsIn a three-way cross-over study we investigated the effects of two doses of RG7203 (5 mg and 15 mg) and placebo given as adjunctive treatment to stable background antipsychotic treatment on reward functioning and reward-based effortful behavior using the monetary incentive delay (MID) task during fMRI and the effort choice task in patients with chronic schizophrenia and moderate levels of negative symptoms (PANSS negative symptom factor score ≥ 18 points). Each treatment period lasted three weeks followed by a 2 week washout period. fMRI and behavioral tasks were administered at the end of each treatment period. Key outcome measures were the differential BOLD during reward anticipation and overall BOLD activity during the MID task and the percentage of high-effort high-reward choices when the probability of reward was 100% during the effort choice task.ResultsThirty-three patients with schizophrenia (30 male; 21 B, 9 W, 3 A; mean age 36.6 ± 7 y; PANSS NSFS = 22.8 (±1.4) at screening) were recruited at three study centers in the US. Twenty-four subjects finished the entire study. RG7203 at 5 mg significantly increased differential BOLD activity during reward anticipation in the MID task. However, this enhancement occurred in the context of a significant decrease of BOLD activity across all conditions during the MID task under treatment with RG7203. RG7203 significantly worsened reward-based effortful behavior in the effort choice task (the high-effort high-reward choice: 67% for both doses of RG7203 versus 73% for placebo). Multiple regression revealed that the decrease in effortful behavior was significantly related to the decrease in overall BOLD activity during the MID task and not related to the difference of BOLD activity during reward anticipation versus the control condition.DiscussionIn contrast to our expectation and previous results in healthy volunteers, RG7203 worsened indices of reward functions which we hypothesize may be due to a further enhancement of D2 antagonistic activity. The results do not support the utility of a PDE10 inhibitor as adjunctive treatment for negative symptoms in patients with schizophrenia. Given the previous observation that RG7203 enhanced reward functions in healthy volunteers who were not treated with D2 antagonist, the results of our study point to potentially deleterious effects of D2 blockade on reward functions and by extension on negative symptoms of schizophrenia. They raise the question if the presence of D2 antagonistic treatment curtails the potential effects of any adjunctive treatment for negative symptoms.

11C](R)-Rolipram positron emission tomography detects DISC1 inhibition of phosphodiesterase type 4 in live Disc1 locus-impaired mice

Although still a matter of controversy, disrupted in schizophrenia protein 1 (DISC1) was suggested as a potential inhibitor of phosphodiesterase 4 (PDE4). We used Disc1 locus impairment (LI) mice to investigate the interaction between PDE4 and DISC 1 in vivo and in vitro. [11C](R)-Rolipram binding was measured by PET in LI (n = 11) and C57BL/6 wild-type (WT, n = 9) mice. [11C](R)-Rolipram total distribution volumes (VT) were calculated and corrected for plasma-free fraction (fP) measured in a separate group of LI (n = 6) and WT (n = 7) mice. PDE4 enzyme activity was measured using in vitro samples of cerebral cortices from groups of LI (n = 4), heterozygote (n = 4), and WT (n = 4) mice. Disc1 LI mice showed a 41% increase in VT (18 ± 6 vs. 13±4 mL/cm3, P = 0.04) compared to WT mice. VT/fP showed a 73% significant increase (90 ± 31 vs. 52 ± 15 mL/cm3, P = 0.004) in Disc1 LI compared to WT mice. PDE4 enzymatic activity assay confirmed in vivo findings showing significant group differences (p < 0.0001). In conclusion, PDE4 activity was increased in the absence of critical DISC1 protein isoforms both in vivo and in vitro. Additionally, [11C](R)-Rolipram PET was sensitive enough to assess altered PDE4 activity caused by PDE4–DISC1 interaction.

Anti-Venom Potential of Canthium parviflorum Against Naja naja Venom by In Vitro and In Vivo Studies

Plant extracts are extensively used in folk medicine to treat the snake bites. In the present study, anti-venom potential of Canthium parviflorum root extracts against Naja naja venom was studied by in vitro and in vivo methods. Ethyl acetate and methanolic root extracts were analysed for in vitro neutralization of 5′-nucleotidase, phospholipase A2, acetylcholinesterase, phosphodiesterase, hyaluronidase, phosphomonoesterase and protease enzyme activities. Neutralization of pharmacological activities like fibrinogenolytic, and direct and indirect haemolytic activities with active methanol extract was performed. Lethal toxicity determination and its inhibition by root extract were carried out in vitro on chick embryo and in vivo on mice model. Further, venom induced edema, myotoxicity and its neutralization were carried out in the mice model. Both extracts inhibited all enzyme activities in a dose dependent manner with various IC50 values. However, only methanol extract effectively neutralized protease activity. Active methanol extract significantly neutralized all the pharmacological activities at various concentrations. Lethal dose (LD50) of venom was 2.5 µg/egg and effective dose of plant was 0.79 mg/egg for 2 LD50 of the venom. LD50 value of venom was 0.38 mg/kg body weight of mice and survival time was prolonged due to plant extract. At 1:10 and 1:20 of venom: plant extract concentration (w/w) significantly neutralized edema and myotoxic effects respectively in mice model. The present study revealed the potential of C. parviflorum root extract for its anti-venom properties and the plants could be considered as a potential source of anti-venom phytoconstituents.

In vivo Analysis of Cyclic di-GMP Cyclase and Phosphodiesterase Activity in Escherichia coli Using a Vc2 Riboswitch-based Assay.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that regulates distinct aspects of bacterial physiology. It is synthesized by diguanylate cyclases (DGCs) and hydrolyzed by phosphodiesterases (PDEs). To date, the activities of DGC and PDE are commonly assessed by phenotypic assays, mass spectrometry analysis of intracellular c-di-GMP concentration, or riboswitch-based fluorescent biosensors. However, some of these methods require cutting-edge equipment, which might not be available in every laboratory. Here, we report a new simple, convenient and cost-effective system to assess the function of DGCs and PDEs in E. coli. This system utilizes the high specificity of a riboswitch to c-di-GMP and its ability to regulate the expression of a downstream β-galactosidase reporter gene in response to c-di-GMP concentrations. In this protocol, we delineate the construction of this system and its use to assess the activity of DGC and PDE enzymes.

Mitochondrial proteomics investigation of frontal cortex in an animal model of depression: Focus on chronic antidepressant drugs treatment.

Alteration in the brain mitochondrial functions have been suggested to participate, as a relevant factor, in the development of mental disorders. Therefore, the brain mitochondria may be a crucial therapeutic target in the course of depression. Our goal was to find out the impact of two antidepressant drugs with various mechanisms of action - imipramine and fluoxetine, on the frontal cortex mitochondria-enriched fraction in an animal model of depression based on the prenatal stress procedure. Our results confirmed that the prenatal stress caused depressive-like disturbances in the adult offspring rats, which were normalized by the chronic imipramine and fluoxetine administration. For the first time, using 2D-LC-MS/MS, we demonstrated nine differentially expressed proteins after the imipramine administration. Of these proteins, the up-regulation of the 2',3'-cyclic-nucleotide 3'-phosphodiesterase enzyme and down-regulation of the Hypoxanthine-guanine phosphoribosyltransferase (HPRT), Ras-related proteins (Rap-1A and Rap-1B) and Transgelin-3 (NP25) were the most striking. In contrast, after the chronic fluoxetine treatment, we observed differential expression in five proteins, including the enhanced expression of component of pyruvate dehydrogenase complex and diminished of Glutathione S-transferase P (Gstp-1), as well as Maleylacetoacetate isomerase. These results overcome the interesting data that brain mitochondria in the frontal cortex may constitute the target for pharmacotherapy. The multifaceted profile of both antidepressant drugs action makes difficult to elucidate the exact mechanism of imipramine and fluoxetine action in the brain mitochondria. Further study of mitochondrial dysfunction in psychiatric disorders will be base to know the possible biological consequences of our observations.

Heptamethoxyflavone Reduces Phosphodiesterase Activity and T-Cell Growth in vitro

In a previous study, we reported that interleukin-4 production was reduced in spleen cells of mice administered 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), which is a polymethoxyflavone found at high concentrations in the peel of various citrus fruits. In this study, we investigated the function of HMF on the growth of T cells cultured from the spleens of mice. HMF decreased the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by anti-CD3/CD28 antibody-stimulated mouse spleen cells. HMF inhibited the activities of phosphodiesterase (PDE) enzymes prepared from bovine brain and human PDE_4B and PDE_3B enzymes. The cyclic AMP (cAMP) content in anti-CD3/CD28 antibody-stimulated spleen cells increased after HMF treatment in vitro. These results suggest that HMF inhibits T-cell growth and affects immune function via reduced PDE activity and increased cAMP content.

Repositioning of molsidomine for its efficacy in diabetes induced erectile dysfunction in rats: In silico, in vitro and in vivo approach.

Well known risk factors for diabetic erectile dysfunction include impaired nitric oxide synthesis and endothelial dysfunction. We proposed to evaluate the efficacy of nitric oxide donor, molsidomine in rat model of diabetic erectile dysfunction. Streptozotocin (52mg/kg, ip) induced diabetic male rats were treated with molsidomine (5 and 10mg/kg, po) for 8 weeks. The sexual behaviour of male rat in presence of the female rat in oestrous phase was observed at the end of study. The effect of treatment on serum testosterone level, sperm parameters and penile tissue histopathology was also evaluated. Further anti-inflammatory activity and antioxidant potential of molsidomine was evaluated by in vitro method. In silico docking study was carried out to appreciate binding conformation of the molsidomine to its plausible target, phosphodiesterase enzyme. Molsidomine significantly and dose dependently increased sexual behaviour, sperm count and serum testosterone level in diabetic rats. Further, the protective effect of molsidomine was also substantiated by pathological changes in the architect of the penile tissue. Molsidomine showed good membrane stability accounting for its significant anti-inflammatory action and also significantly scavenged DPPH radical activity showing its antioxidant action. Molsidomine was found to settle well in the active site of PDE-5 enzyme with less binding affinity than the standard drug sildenafil. The results highlight the rationale behind the repositioning of molsidomine therapy for the management of diabetic erectile dysfunction.

Targeting PDE10A GAF Domain with Small Molecules: A Way for Allosteric Modulation with Anti-Inflammatory Effects.

Phosphodiesterase (PDE) enzymes regulate the levels of cyclic nucleotides, cAMP, and/or cGMP, being attractive therapeutic targets. In order to modulate PDE activity in a selective way, we focused our efforts on the search of allosteric modulators. Based on the crystal structure of the PDE10A GAF-B domain, a virtual screening study allowed the discovery of new hits that were also tested experimentally, showing inhibitory activities in the micromolar range. Moreover, these new PDE10A inhibitors were able to decrease the nitrite production in LPS-stimulated cells, thus demonstrating their potential as anti-inflammatory agents.

PDE8 controls CD4+ T cell motility through the PDE8A-Raf-1 kinase signaling complex

The levels of cAMP are regulated by phosphodiesterase enzymes (PDEs), which are targets for the treatment of inflammatory disorders. We have previously shown that PDE8 regulates T cell motility. Here, for the first time, we report that PDE8A exerts part of its control of T cell function through the V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase signaling pathway. To examine T cell motility under physiologic conditions, we analyzed T cell interactions with endothelial cells and ligands in flow assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 suppresses adhesion of in vivo myelin oligodendrocyte glycoprotein (MOG) activated inflammatory CD4+ T effector (Teff) cells to brain endothelial cells under shear stress. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP levels around Raf-1 thereby protecting it from protein kinase A (PKA) mediated inhibitory phosphorylation. To test the function of this complex in Teff cells, we used a cell permeable peptide that selectively disrupts the PDE8A-Raf-1 interaction. The disruptor peptide inhibits the Teff–endothelial cell interaction more potently than the enzymatic inhibitor. Furthermore, the LFA-1/ICAM-1 interaction was identified as a target of disruptor peptide mediated reduction of adhesion, spreading and locomotion of Teff cells under flow. Mechanistically, we observed that disruption of the PDE8A-Raf-1 complex profoundly alters Raf-1 signaling in Teff cells. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase complex disruptors decreases Teff cell adhesion and migration under flow, and represents a novel approach to target T cells in inflammation.

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling.

Many aspects of bacterial physiology and behavior, including motility, surface attachment, and the cell cycle, are controlled by cyclic di-GMP (c-di-GMP)-dependent signaling pathways on the scale of seconds to minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing the expression of genes encoding c-di-GMP-synthetic (diguanylate cyclases) and -degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared-light-regulated diguanylate cyclase, BphS, was engineered previously, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue-light-activated phosphodiesterase EB1 can be used in combination with the red/near-infrared-light-regulated diguanylate cyclase BphS for the bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility, attachment to surfaces, the cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occur at a fast (seconds-to-minutes) pace. Interrogation of these processes at high temporal and spatial resolution using chemicals is difficult or impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue-light-activated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared-light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.

Effect of pineapple, orange and watermelon juices on phosphodiesterase, monoamine oxidase and angiotensin-I converting enzyme activities in rat heart and brain homogenates

Fruit juices are commonly consumed in many parts of the world for their nutritional and medicinal benefits. More specifically, Pineapple (Ananas comosus), orange (Citrus sinensis) and watermelon (Citrullus lanatus var. lanatus) juices are used in folklore for the prevention and management of cardiovascular and neurodegenerative diseases, though there is dearth of information on the mechanism for such medicinal benefits. Thetrefore, this study sought to characterize the effect of these commonly consumed fruit juices on phosphodiesterase (PDE), monoamine oxidase (MAO) and angiotensin-I converting enzyme (ACE) activities in rat heart and brain homogenates. The fruits were sourced for and the juices extracted, freeze dried and then reconstituted for the analysis. The abilities of these juices to protect the heart and brain against Fe2+ induced oxidative damage were also investigated in vitro. The results revealed that the juices inhibited PDE, MAO and ACE activities in a concentration-dependent manner in rat heart and brain homogenates with orange having the highest inhibitory ability. The juices also inhibited Fe2+ induced malondialdehyde production in rat heart and brain in a concentration-dependent manner. This study revealed that the inhibition of these enzymes and prevention of oxidative damage could be part of the mechanisms by which these fruits could serve as dietary intervention for the prevention and management of cardiovascular and neurodegenerative diseases.

Discovery of Two Bacterial Nitric Oxide-Responsive Proteins and Their Roles in Bacterial Biofilm Regulation

Bacterial biofilms form when bacteria adhere to a surface and produce an exopolysaccharide matrix ( Costerton Science 1999 , 284 , 1318 ; Davies Science 1998 , 280 , 295 ; Flemming Nat. Rev. Microbiol. 2010 , 8 , 623 ). Because biofilms are resistant to antibiotics, they are problematic in many aspects of human health and welfare, causing, for instance, persistent fouling of medical implants such as catheters and artificial joints ( Brunetto Chimia 2008 , 62 , 249 ). They are responsible for chronic infections in the lungs of cystic fibrosis patients and in open wounds, such as those associated with burns and diabetes. They are also a major contributor to hospital-acquired infections ( Sievert Infec. Control Hosp. Epidemiol. 2013 , 34 , 1 ; Tatterson Front. Biosci. 2001 , 6 , D890 ). It has been hypothesized that effective methods of biofilm control will have widespread application ( Landini Appl. Microbiol. Biotechnol. 2010 , 86 , 813 ). A promising strategy is to target the mechanisms that drive biofilm dispersal, because dispersal results in biofilm removal and in the restoration of antibiotic sensitivity. First documented in Nitrosomonas europaea ( Schmidt J. Bacteriol. 2004 , 186 , 2781 ) and the cystic fibrosis-associated pathogen Pseudomonas aeruginosa ( Barraud J. Bacteriol. 2006 , 188 , 7344 ; J. Bacteriol. 2009 , 191 , 7333 ), regulation of biofilm formation by nanomolar levels of the diatomic gas nitric oxide (NO) has now been documented in numerous bacteria ( Barraud Microb. Biotechnol. 2009 , 2 , 370 ; McDougald Nat. Rev. Microbiol. 2012 , 10 , 39 ; Arora Biochemistry 2015 , 54 , 3717 ; Barraud Curr. Pharm. Des. 2015 , 21 , 31 ). NO-mediated pathways are, therefore, promising candidates for biofilm regulation. Characterization of the NO sensors and NO-regulated signaling pathways should allow for rational manipulation of these pathways for therapeutic applications. Several laboratories, including our own, have shown that a class of NO sensors called H-NOX (heme-nitric oxide or oxygen binding domain) affects biofilm formation by regulating intracellular cyclic di-GMP concentrations and quorum sensing ( Arora Biochemistry 2015 , 54 , 3717 ; Plate Trends Biochem. Sci. 2013 , 38 , 566 ; Nisbett Biochemistry 2016 , 55 , 4873 ). Many bacteria that respond to NO do not encode an hnoX gene, however. My laboratory has now discovered an additional family of bacterial NO sensors, called NosP (nitric oxide sensing protein). Importantly, NosP domains are widely conserved in bacteria, especially Gram-negative bacteria, where they are encoded as fusions with or in close chromosomal proximity to histidine kinases or cyclic di-GMP synthesis or phosphodiesterase enzyme, consistent with signaling. In this Account, we briefly review NO and H-NOX signaling in bacterial biofilms, describe our discovery of the NosP family, and provide support for its role in biofilm regulation in Pseudomonas aeruginosa, Vibrio cholerae, Legionella pneumophila, and Shewanella oneidensis.

Phosphorus mobilization in the Yeyahu Wetland: Phosphatase enzyme activities and organic phosphorus fractions in the rhizosphere soils

Phosphatase enzymes regulate organic phosphorus (OP) turnover in eutrophic wetland soils, but a clear understanding of their relationships remains elusive. In this study, soil samples were collected from rhizosphere and non-rhizosphere soils of three dominant aquatic plants Phragmites communis, Zizania latifolia, and Scirpus planiculmis in Yeyahu Wetland of Beijing, China to investigate the phosphatase enzyme activities, including phosphomonoesterase, phosphodiesterase and phytase, and the impact on OP turnover dynamics in wetlands. Results demonstrated that soil phosphatase activities in rhizosphere were higher than those in non-rhizosphere for all the three plants. Phosphatase activities were correlated strongly with soil labile organic P, although the relationships showed different trends for the three kinds of phosphatases. Moderate liable organic P had a positive correlation to phosphomonoesterase (r = 0.367, P < 0.01) and phosphodiesterase (r = 0.564, P < 0.01). There was a significant negative correlation between highly resistant organic P and phytase (r = −0.438, P < 0.05), indicating that phytase had obvious effects on the process of degrading highly stable P compounds in soils. These results suggest that phosphatase enzymes regulated OP turnover to increase available P and phytase had a strong impact on wetland P cycle system.

Therapeutic Targeting of PDEs and PI3K in Heart Failure with Preserved Ejection Fraction (HFpEF).

Heart Failure with preserved Ejection Fraction (HFpEF) is a prevalent disease with considerable individual and societal burden. HFpEF patients often suffer from multiple pathological conditions thatcomplicate management and adversely affect outcome, including pulmonary hypertension and chronic obstructive pulmonary disease (COPD). To date, no treatment proved to be fully effective in reducing morbidity and mortality in HFpEF, possibly due to an incomplete understanding of the underlying molecular mechanisms. The emerging view proposes chronic systemic inflammation, leading to endothelial dysfunction and interstitial fibrosis, as a prominent cause of HFpEF, rather than a mere co-existent disease. In the last decade, efforts from pharmaceutical companies attempted to target pharmacologically enzymes which play key roles in systemic and lung inflammation, such as the cyclic nucleotide-degrading enzymes phosphodiesterases (PDEs) and phosphoinositide-3 phosphate kinases (PI3Ks), especially to limit COPD. In this review, we will summarize major successes and drawbacks of hitting these enzymes to tackle inflammation in HFpEF-associated co-morbidities, with a major focus on the results of completed and ongoing clinical trials. Finally, we will discuss the potential of repurposing and/or developing new PDE and PI3K inhibitors for HFpEF therapy.

Enzymatic activity of the CaM-PDE1 system upon addition of actinyl ions

The threat of a dirty bomb which could cause internal contamination has been of major concern for the past decades. Because of their high chemical toxicity and their presence in the nuclear fuel cycle, uranium and neptunium are two actinides of high interest. Calmodulin (CaM) which is a ubiquitous protein present in all eukaryotic cells and is involved in calcium-dependent signaling pathways has a known affinity for uranyl and neptunyl ions. The impact of the complexation of these actinides on the physiological response of the protein remains, however, largely unknown. An isothermal titration calorimetry (ITC) was developed to monitor in vitro the enzymatic activity of the phosphodiesterase enzyme which is known to be activated by CaM and calcium. This approach showed that addition of actinyl ions (AnO2n+), uranyl (UO22+) and neptunyl (NpO2+), resulted in a decrease of the enzymatic activity, due to the formation of CaM-actinide complexes, which inhibit the enzyme and alter its interaction with the substrate by direct interaction. Results from dynamic light scattering rationalized this result by showing that the CaM-actinyl complexes adopted a specific conformation different from that of the CaM-Ca2+ complex. The effect of actinides could be reversed using a hydroxypyridonate actinide decorporation agent (5-LIO(Me-3,2-HOPO)) in the experimental medium demonstrating its capacity to efficiently bind the actinides and restore the calcium-dependent enzyme activation.