Phosphodiesterase Activity Research Articles

Pharmacological Profile of Difamilast, a Novel Selective Phosphodiesterase 4 Inhibitor, for Topical Treatment of Atopic Dermatitis.

PDE4 inhibitors are expected to be anti-inflammatory agents based on their mechanism of action, but the application of this drug class is limited by a narrow therapeutic window due to adverse effects associated with gastrointestinal function. Difamilast, a novel selective phosphodiesterase 4 (PDE4) inhibitor, demonstrated significant efficacy without adverse reactions such as nausea and diarrhea in patients with atopic dermatitis (AD) and was recently approved in Japan. In this study, we investigated the pharmacological and pharmacokinetic properties of difamilast to provide nonclinical data to help understand the clinical effects. Difamilast selectively inhibited recombinant human PDE4 activity in assays. The IC50 of difamilast against PDE4B, a PDE4 subtype that plays an important role in the inflammatory response, was 0.0112 μM, representing a 6.6-fold decrease compared with the IC50 against PDE4D (0.0738 μM), a subtype that can trigger emesis. Difamilast inhibited TNF-α production in human (IC50 = 0.0109 μM) and mouse (IC50 = 0.0035 μM) peripheral blood mononuclear cells and improved skin inflammation in a mouse model of chronic allergic contact dermatitis. These effects of difamilast on TNF-α production and dermatitis were superior to those of other topical PDE4 inhibitors: CP-80633, cipamfylline, and crisaborole. In pharmacokinetic studies using miniature pigs and rats, the concentrations of difamilast in the blood and brain after topical application were not sufficient to support pharmacological activity. This nonclinical study contributes to explain the efficacy and safety of difamilast with a sufficient therapeutic window in the clinical trials. SIGNIFICANCE STATEMENT: This is the first report on the nonclinical pharmacological profile of difamilast ointment, a novel topical PDE4 inhibitor that demonstrated utility in clinical trials in patients with atopic dermatitis. Difamilast, which has high PDE4 selectivity (especially for the PDE4B subtype), ameliorated chronic allergic contact dermatitis in mice after topical application, with a pharmacokinetic profile in animals that suggests few systemic side effects; thus, difamilast is a promising new therapeutic treatment for atopic dermatitis.

Effect of the Hydroalcoholic Extract from the Leafy Stems of Waltheria indica L. (Malvaceae) on Acetylcholine and Barium Chloride-Induced Contractions on Isolated Rat Tracheal Tissue

A previous study has reported the interesting relaxant effect of the hydroalcoholic extract from Waltheria indica L. (Malvaceae) leafy stems, a plant with several therapeutic uses. The present study aimed to investigate the preventive effect of this plant using an ex vivo model of the rat trachea. Two agonists, acetylcholine (10-6 - 1.5×10-5 M) and barium chloride (10-5- 10-1 M) were used to induce contractions. The preventive effect was assessed on rat tracheal rings pretreated with hydroalcoholic extract (1.92 mg/mL), glibenclamide, atropine, and papaverine, all at 10 µM. Acetylcholine and barium chloride provoke contractions in a concentration-dependent manner, with a maximum contractile response of 3.953 ± 0.692 g and 2.999 ± 0.326 g, respectively. The EC50 values were 3.711 ± 0.823 µM and 9.502 ± 12.354 mM, respectively, for acetylcholine- and barium chloride-induced contraction. Glibenclamide caused a rightward shift of the acetylcholine-response curve, followed by a reduction of the maximum contraction (from 3.953 ± 0.692 g to 3. 116 ± 0.244 g). The hydroalcoholic extract, atropine, papaverine, and their combinations induced a complete suppression of the contractile response to acetylcholine (p < 0.0001) and barium chloride (p < 0.0001). The hydroalcoholic extract exhibited a potent relaxant effect comparable to that of atropine and papaverine. It can be concluded that the hydroalcoholic extract of W. indica can potently prevent acetylcholine-and barium chloride-induced contractions. The possible mechanisms by which the extract exerts its relaxant effect may involve the blockade of muscarinic receptors, the inhibition of phosphodiesterase activity, and/or the calcium channel.

Narrative Review of the Pathogenesis of Stasis Dermatitis: An Inflammatory Skin Manifestation of Venous Hypertension.

Stasis dermatitis (SD), also known as venous dermatitis, is a form of inflammatory dermatitis of the lower extremities that typically occurs in older individuals and represents a cutaneous manifestation of venous hypertension. Venous hypertension (also known as sustained ambulatory venous pressure) is most often due to retrograde blood flow, which occurs due to calf muscle pump failure. This failure is most commonly secondary to incompetent venous valves, valve destruction, or obstruction of the venous system. Many of the common symptoms associated with SD are caused by inflammatory processes. This review summarizes the pathogenesis and key role of inflammation in SD by reviewing inflammatory biomarkers associated with SD. The literature was selected though a high-level PubMed search focusing on keywords relating to inflammation associated with SD. Venous reflux at the lower extremities causes venous hypertension, which leads to chronic venous insufficiency. High venous pressure due to venous hypertension promotes the local accumulation and extravasation of inflammatory cells across the vascular endothelium. Leukocyte trapping in the microcirculation and perivascular space is associated with trophic skin changes. Cell adhesion molecules are linked with the perpetuated influx of activated leukocytes into inflammatory sites. Here, inflammatory cells may influence the remodeling of the extracellular matrix by inducing the secretion of proteinases such as matrix metalloproteinases (MMPs). The increased expression of MMPs is associated with the formation of venous leg ulcers and lesions. Phosphodiesterase4 activity has also been shown to be elevated in individuals with inflammatory dermatoses compared to healthy individuals. Because inflammation is a key driver of the signs and symptoms of SD, several of the highlighted biomarkers of inflammation represent potential opportunities to target and interrupt molecular pathways of cutaneous inflammation and, therefore, remediate the signs and symptoms of SD. Understanding the pathogenesis of SD may help clinicians identify drivers of inflammation to use as potential targets for the development of new treatment options.

Phosphorus Use Efficiency of Leafy Brassica sp. Grown in Three Contrasting Soils: Growth, Enzyme Activity and Phosphorus Fractionation

Plant adaptations to low soil phosphorus (P) availability have been intensively studied in Brassica sp. in an attempt to identify the mechanisms involved in P uptake and utilization. The present pot experiment was conducted to evaluate the relationships between plant shoot and root growth, P uptake and use efficiency parameters, and P fractions and enzyme activity, in two species grown in three soil types. The aim of this study was to determine whether adaptation mechanisms are soil-dependent. Two kale species were grown in soils typical for coastal Croatia (terra rossa, rendzina, and fluvisol) with low P availability. Plants grown in fluvisol had the highest shoot biomass and accumulated most P, whereas plants developed the longest roots in terra rossa. Phosphatase activity differed among soils. P use efficiency differed among soils and species. Genotype IJK 17 showed better adaptation to low P availability, which was related to better uptake efficiency. In general, soils differed in inorganic and organic P fractions in rhizosphere soil, but no difference between genotypes was found. The activities of alkaline phosphatase and phosphodiesterase were negatively correlated with most organic P fractions, suggesting their function in the mineralization of soil organic P. Kale species activate different mechanisms of P uptake and utilization when grown in contrasting soil types, suggesting that specific responses to the soil type were more important than the genotypic difference.

Vertical changes in P-acquiring enzyme activities and microbial biomass in Luvisols – The effect of different types of agricultural land use and soil-forming processes

The activities of enzymes involved in the transformation of soil organic P are rarely measured in soils below a 30 cm depth, even though substantial amounts of P are also stored in deep soils. Changes in soil microbial biomass and the activities of P-cycling enzymes with depth were determined across the eight Luvisol profiles in four agricultural land uses including lucerne, winter wheat, vineyards and orchards. The activities of acid phosphatase (AcP), alkaline phosphatase (AlP) and phosphodiesterase (PDE) were assessed, as was the microbial biomass carbon (MBC) and phosphorus content (MBP) as well as some physico-chemical properties (e.g. total organic carbon – TOC, total nitrogen – TN, available phosphorus – Pavail, bulk density, pH in CaCl2). Additionally, mass and morphological parameters of the plant roots were also determined. The highest enzymatic activity and microbial biomass C and P content were found in the surface and sub-surface horizons and decreased with depth to varying degrees in parallel to the changes in TOC and TN. The studied properties were primarily determined by means of the organic C content and availability in all genetic horizons, while the influence of the lessivage (clay content) was only clearly pronounced for AcP activity, which was significantly higher in the illuvial layers (E) as compared to the eluvial horizons (Bt) in most of the considered profiles. What is more, only AcP activity was significantly and positively related to the root biomass and the number of small roots in the plants. Although it was revealed that the particular plants being cultivated had a significant impact on AcP activity, the data should be interpreted with caution because other factors (e.g. mineral fertilization) may also contribute to this.

Inhibiting bacterial biofilm formation by stimulating c-di-GMP regulation using citrus peel extract from Jeju Island

Biofilms consist of single or multiple species of bacteria embedded in extracellular polymeric substances (EPSs), which affect the increase in antibiotic resistance by restricting the transport of antibiotics to the bacterial cells. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In this study, we found that citrus peel extract from Jeju Island (CPEJ) can inhibit bacterial biofilm formation. According to the results, CPEJ concentration-dependently reduces biofilm formation without affecting bacterial growth. Additionally, CPEJ decreased the production of extracellular polymeric substances but increased bacterial swarming motility. These results led to the hypothesis that CPEJ can reduce intracellular bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) concentration. The results showed that CPEJ significantly reduced the c-di-GMP level through increased phosphodiesterase activity. Altogether, these findings suggest that CPEJ as a biofilm inhibitor has new potential for pharmacological (e.g. drug and medication) and industrial applications (e.g. ship hulls, water pipes, and membrane processes biofouling control).

H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains (avi_3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that avH-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi_3097 (avHaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of avHaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound avH-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant ΔhnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis. Together, these data indicate that avH-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis.

Role of mitochondrial pka in compartmentation of cAMP signaling in cardiac ventricular myocytes.

In cardiac ventricular myocytes, both β1 and β2-adrenergic receptors (βARs) produce an increase in the rate and force of contraction by stimulating cAMP production. However, only β1ARs enhance the rate of relaxation by regulating the activity of the SERCA2 pump found in the distal scaroplasmic reticulum. Previously we found that β1, but not β2AR production of cAMP could be detected by Epac2-αKAP, a FRET-based biosensor targeted to the the SERCA2 signaling complex, demonstrating that compartmentation of cAMP signaling contributes to the difference in functional responses. While inhibition of phosphodiesterase (PDE) activity was found to influence this behavior, in silico studies have suggested that intracellular cAMP must also be slowed by other mechanisms in order for this to occur. Consistent with this prediction, using raster image correlation spectroscopy (RICS), we found that the intracellular diffusion coefficient of fluorescently labeled cAMP is actually 30 fold slower than the rate of free diffusion. Furthermore, this slowing of diffusion was found to be due to the buffering effect of protein kinase A (PKA), specifically PKA anchored to the outer membrane of mitochondria. To directly test the hypothesis that this buffering effect contributes to cAMP compartmentation, we used adenoviral-mediated knockdown to target two key mitochondrial A kinase anchoring proteins (AKAPs), D-AKAP1 and D-AKAP2. Following knockdown of either AKAP alone, we found that the cAMP diffusion coefficient was significantly increased compared to control. Furthermore, knocking down the expression of D-AKAP1 enhanced β2AR stimulation of cAMP production detected by Epac2-αKAP. These results support the conclusion that buffering of cAMP mobility by PKA anchored to mitochondria influences cAMP dynamics and contributes to compartmentation of β2AR signaling in cardiac ventricular myocytes.

Prune regulates the metabolism of mammalian inorganic polyphosphates and bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved, ubiquitous polymer that is present in all studied organisms. It has shown a preferred localization within the mitochondria and it is composed of multiple orthophosphates (Pi) linked together by high energy phosphoanhydride bonds, identical to those found in ATP. The metabolism of polyP is well described in bacteria and yeast, but has yet to be elucidated in higher eukaryotes. However, recent evidence suggests that the mammalian F0F1 ATP synthase could be involved in both the synthesis and hydrolysis of polyP. The mitochondrial localized protein h-Prune, the human ortholog of Drosophila Prune, exhibits short chain exopolyphosphatase activity in vitro, in addition to its known cAMP phosphodiesterase activity. Here, we studied the role of prune orthologs in polyP metabolism in both mammalian HEK293 cells and Drosophila melanogaster. Our data shows that purified h-Prune is unable to hydrolyze polyP with chain lengths spanning from 13-33 Pi long. We then used interfering RNA to knock down the expression of prune orthologs and found that the polyP levels were significantly reduced in both HEK293 and Drosophila melanogaster. In HEK293 cells, ATP levels were also significantly decreased by h-Prune knockdown, although mitochondrial membrane potential was not affected. This suggests that h-Prune could either directly or indirectly influence the activity of the F0F1 ATP synthase. Concordantly, we found that h-Prune knockdown significantly decreased ATP synthase activity to levels comparable to inhibition by Oligomycin A. Our results collectively demonstrate the downstream ability of prune to regulate cellular polyP levels and bioenergetics, although further studies are required to elucidate the exact mechanism of this pathway.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological β-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

Polyphenol-Rich Beverage Consumption Affecting Parameters of the Lipid Metabolism in Healthy Subjects.

Polyphenols are a diverse and widely distributed class of secondary metabolites, which possess numerous beneficial properties including a modulation of glucose and lipid metabolism. This placebo-controlled human intervention study was performed to explore effects of polyphenol-rich beverage (PRB) uptake on lipid metabolism, as well as DNA integrity. In this case, 36 healthy men were randomly divided to consume either 750 mL of a PRB (containing 51% chokeberry, cranberry, and pomegranate) or a placebo drink daily for eight weeks. Only PRB consumption was found to decrease fat and protein intakes significantly compared to the preceding one-week washout period. During the intervention with PRB an increased fat-free mass was shown after four weeks, whereas a significant elevation in body weight and leptin was observed in placebo group. Blood lipids were not significantly altered after PRB consumption, while triglyceride levels increased after placebo drink intake. In platelets, a significant inhibition of phosphodiesterase (PDE) activity was observed, more pronounced in test group. Consuming the PRB decreased total DNA strand breaks in whole blood as well as H2O2-induced breaks in isolated lymphocytes. Overall, our study suggested beneficial effects on lipid metabolism by reduced energy intake, modulation of biomarkers such as PDE activity and improved DNA integrity associated with PRB consumption.

Crisaborole efficacy in murine models of skin inflammation and Staphylococcus aureus infection.

Phosphodiesterase 4 (PDE4) is highly expressed in keratinocytes and immune cells and promotes pro-inflammatory responses upon activation. The activity of PDE4 has been attributed to various inflammatory conditions, leading to the development and approval of PDE4 inhibitors as host-directed therapeutics in humans. For example, the topical PDE4 inhibitor, crisaborole, is approved for the treatment of mild-to-moderate atopic dermatitis and has shown efficacy in patients with psoriasis. However, the role of crisaborole in regulating the immunopathogenesis of inflammatory skin diseases and infection is not entirely known. Therefore, we evaluated the effects of crisaborole in multiple mouse models, including psoriasis-like dermatitis, AD-like skin inflammation with and without filaggrin mutations, and Staphylococcus aureus skin infection. We discovered that crisaborole dampens myeloid cells and itch in the skin during psoriasis-like dermatitis. Furthermore, crisaborole was effective in reducing skin inflammation in the context of filaggrin deficiency. Importantly, crisaborole reduced S. aureus skin colonization during AD-like skin inflammation. However, crisaborole was not efficacious in treating S. aureus skin infections, even as adjunctive therapy to antibiotics. Taken together, we found that crisaborole reduced itch during psoriasis-like dermatitis and decreased S. aureus skin colonization upon AD-like skin inflammation, which act as additional mechanisms by which crisaborole dampens the immunopathogenesis in mouse models of inflammatory skin diseases. Further examination is warranted to translate these preclinical findings to human disease.

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The β-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

Depth-Related Changes in Soil P-Acquiring Enzyme Activities and Microbial Biomass—The Effect of Agricultural Land Use/Plant Cover and Pedogenic Processes

Although the phosphatase enzymes regulate phosphorus (P) turnover throughout the soil profile, at present, they are rarely studied and are less well understood in the deeper soil layers than in the surface horizons. Hence the changes in P-associated soil properties were assessed throughout five Phaeozem profiles for different agricultural land uses including alfalfa, winter wheat, grapevine, apple trees and hops. The acid phosphatase (AcP), alkaline phosphatase (AlP) and phosphodiesterase (PDE) activity was assayed, as were the microbial biomass carbon (MBC) and phosphorus (MBP) contents and also other properties (e.g., available phosphorus, total organic carbon, total nitrogen). We have also determined the mass, length and surface area of the plant roots. In general, the activities of the studied enzymes were highest in the topsoil in four out of the five profiles studied, which corresponded to the highest level of root mass. The older the plant, the greater the root mass and increased enzymatic activity in the deeper horizons of apple trees and hop profiles in comparison to the surface layers. The greatest enzymatic activity, MBC and MBP contents were found in the horizons with a TOC content >0.5% and decreased down the soil profiles similarly to the changes in TOC and TN contents. While the studied properties were determined to varying degrees by means of the organic C content and availability in all of the genetic horizons, the influence of the prevailing conditions and the factors related to soil depth and pedogenic processes were less pronounced. The clay content was related to a significant extent to all of the studied enzyme activities, but only in horizons with a TOC content <0.5%. Significantly higher phosphatase activity under aerobic as opposed to anaerobic conditions were determined in this study, while the opposite trend was found for the content of MBC and MBP as well as the ratio of MBC/MBP. Overall, we pointed out the complex effect of the soil depth, soil forming-processes and cultivated plants on soil P-associated enzyme activities and other properties throughout the soil profiles. This knowledge will allow better understanding of the state of enzymes and their contribution to the biogeochemical cycle of soil P, especially in subsoils, where the enzyme activities follow different patterns than those in the surface horizons.

Levosimendan increases the phosphorylation state of phospholamban in the isolated human atrium

Levosimendan (up to 10 µM) given alone failed to increase force of contraction in isolated electrically stimulated (1 Hz) left atrial (LA) preparations from wild-type mice. Only in the additional presence of 0.1 µM rolipram, an inhibitor of the activity of phosphodiesterase IV, levosimendan increased force of contraction in LA and increased the phosphorylation state of phospholamban at amino acid serine 16. Levosimendan alone increased the beating rate in isolated spontaneously beating right atrial preparations from mice and this effect was potentiated by rolipram. The positive inotropic and the positive chronotropic effects of levosimendan in mouse atrial preparations were attenuated by 10 µM propranolol. Finally, we studied the contractile effects of levosimendan in isolated electrically stimulated (1 Hz) right atrial preparations from the human atrium (HAP), obtained during cardiac surgery. We detected concentration-dependent positive inotropic effects of levosimendan alone that reached plateau at 1 µM levosimendan in HAP (n = 11). Levosimendan shortened time of tension relaxation in HAP. Cilostamide (1 µM), an inhibitor of phosphodiesterase III, or propranolol (10 µM) blocked the positive inotropic effect of levosimendan in HAP. Levosimendan (1 µM) alone increased in HAP the phosphorylation state of phospholamban. In conclusion, we present evidence that levosimendan acts via phosphodiesterase III inhibition in the human atrium leading to phospholamban phosphorylation and thus explaining the positive inotropic effects of levosimendan in HAP.

Kinetic Features of 3'-5'-Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae.

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5' side of an AP site, thereby forming a single-strand break containing 3'-OH and 5'-dRP ends. In addition, Apn2 has 3'-phosphodiesterase activity (removing 3'-blocking groups) and 3' → 5' exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3'-5'-exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single-strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped-flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5' overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate-limiting step in the enzymatic reaction. We determined an influence of the nature of the 3'-terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context-specific.

Maize-soybean intercropping facilitates chemical and microbial transformations of phosphorus fractions in a calcareous soil.

Intercropping often substantially increases phosphorus (P) availability to plants compared with monocropping, which could be an effective strategy for soil legacy P recovery and agricultural production. However, the biogeochemical interactions among plants, microbes, and soil that mobilize P remain largely unknown in intercropping systems. Pot experiments with maize-soybean intercropping in a calcareous soil were conducted to investigate the potential chemical and biological transformation mechanisms of inorganic P (Pi) and organic P (Po) using sequential extraction and Illumina MiSeq sequencing. Compared to monocropping of each crop, maize-soybean intercropping significantly enhanced total P uptake of the two crops by mobilizing Ca2-Pi [extracted by bicarbonate (NaHCO3)], Al-Pi/Po [extracted by ammonium fluoride (NH4F)] and Fe-Pi [extracted by sodium hydroxide and sodium carbonate (NaOH-Na2CO3)] fractions. Furthermore, there were significant increases in the organic carbon content and alkaline phosphomonoesterase (ALP) and phosphodiesterase (PDE) activities as well as the abundances of Microvirga, Lysobacter, Microlunatus and Sphingomonas under maize-soybean intercropping relative to monocropping. In contrast, compared to monocroppping, no significant change in the soil pH was observed under maize-soybean intercropping. Therefore, the enhanced P uptake of the maize-soybean intercropping probably resulted from a synergistic effect of rhizosphere organic carbon deposit, increased activities of ALP and PDE, together with the bacteria (Microvirga, Lysobacter, Microlunatus and Sphingomonas) which showed correlation with soil P forms, while the generally recognized rhizosphere acidification was excluded in this investigated calcareous soil. Moreover, the selected bacterial genera exhibited a closer network in the rhizosphere of soybean compared to maize, suggesting enhanced interactions among bacteria in the soybean rhizosphere. These results provide theoretical bases for the recovery of soil legacy P by maize-soybean intercropping.

Effects of various straw incorporation strategies on soil phosphorus fractions and transformations

AbstractReturning straw to the field is an effective method for optimizing the soil phosphorus (P) availability, in which bacteria play an important role. However, the effects of various straw incorporation strategies on P transformation between different soil P pools remain unclear. In this study, variations in soil P fractions, phosphatase activities and the abundance of phosphatase genes (phoD, phoX and phoC) as well as a P‐solubilizing gene (pqqC) at DNA (total) and cDNA (transcribed) levels were analysed in three straw incorporation treatments, including chopped straw (StrawD), straw compost (Compost) and straw‐derived biochar (Biochar), and control (no straw, CK). Compared with the CK, the moderately labile inorganic P (NaOH I‐Pi) content significantly decreased and the non‐available P (Residual P) content significantly increased in the StrawD treatment. At the same time, phosphodiesterase (PD) activity and the transcribed phoC and phoX genes as well as total pqqC gene abundance significantly increased in the StrawD treatment, suggesting that the input of chopped straw stimulated P transformations from both organic and inorganic P pools. In addition, the stable Pi (NaOH II‐Pi) content and total pqqC gene abundance in the Biochar treatment were significantly higher than that in the CK, indicating that the input of biochar increased the NaOH II‐Pi that could release available P by Pi‐solubilizing bacteria. In comparison to the CK, the Compost input significantly decreased one labile Pi (resin‐Pi) only. However, its P fractions were significantly different from that of CK, Biochar and StrawD treatments, suggesting that the effects of compost input on P should not be ignored. In conclusion, chopped straw input increased soil P transformation but not available P, biochar input may promote inorganic P transformation, and compost input has a latent effect on P transformation. The study provided a comprehensive understanding of straw incorporation strategies for regulating soil P availability.

A Review Article on Synthesis of Different Types of Bioactive Spiropyrazole Derivatives

Pyrazole category with all derivatives have well-known with biological and industrial applications. Pyrazole combined with other cycle (aliphatic or heterocycles) through one carbon atoms afforded spiropyrazoles. This spiropyrazoles characterized with biological importance as antitumor, analgesic, anti-inflammatory, vasodilation, aldosterone antagonistic, antimicrobial, phosphodiesterase, anabolic, androgenic, progestational, and salt-retaining activities. We interested in this review to collect and sort the spiropyrazoles according to their combined rings and illustrated the different methods of synthesis until the middle of 2021.