Search

Saturated free fatty acid-induced adipocyte inflammation plays a pivotal role in implementing insulin resistance and type 2 diabetes. Recent reports suggest A 2A adenosine receptor (A 2A AR) could be an attractive choice to counteract adipocyte inflammation and insulin resistance. Thus, an effective A 2A AR agonist devoid of any toxicity is highly appealing. Here, we report that indirubin-3'-monoxime (I3M), a derivative of the bisindole alkaloid indirubin, efficiently binds and activates A 2A AR which leads to the attenuation of lipid-induced adipocyte inflammation and insulin resistance. Using a combination of in silico virtual screening of potential anti-diabetic candidates and in vitro study on insulin-resistant model of 3T3-L1 adipocytes, we determined I3M through A 2A AR activation markedly prevents lipid-induced impairment of the insulin signaling pathway in adipocytes without any toxic effects. While I3M restrains lipid-induced adipocyte inflammation by inhibiting NF-κB dependent pro-inflammatory cytokines expression, it also augments cAMP-mediated CREB activation and anti-inflammatory state in adipocytes. However, these attributes were compromised when cells were pretreated with the A 2A AR antagonist, SCH 58261 or siRNA mediated knockdown of A 2A AR. I3M, therefore, could be a valuable option to intervene adipocyte inflammation and thus showing promise for the management of insulin resistance and type 2 diabetes., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The pathophysiological mechanisms of sepsis-induced cardiac dysfunction are largely unknown. The Toll-like receptor 4 (TLR4) is expressed in cardiac myocytes and is involved in bacterial endotoxin-mediated inflammatory disorders. TLR4 signaling leads to activation of the nuclear factor kappa B followed by increased expression of cytokines. Several protein phosphatases including PP2Cβ, PP2A or PP1 are known to act as regulators of this signaling pathway. Here, we examined the role of PP5 for the inflammatory response to the bacterial endotoxin lipopolysaccharide in the heart using a transgenic mouse model with cardiac myocyte directed overexpression of PP5. In these transgenic mice, basal cardiac contractility was reduced, in vivo as well as in vitro, but LPS-induced cardiac dysfunction was less pronounced compared to wild type mice. Quantitative RT-PCR suggested an attenuated NF-κB signaling in the heart and cardiac expression of heat shock protein 25 (HSP25) was increased in PP5 transgenic mice. From our data we assume that PP5 increases stress tolerance of cardiac myocytes by downregulation of NF-κB signaling and upregulation of HSP25 expression., Competing Interests: The authors have declared that no competing interests exist.

Acid-sensing ion channels (ASICs) are proton-gated sodium-selective channels that are expressed in the peripheral and central nervous systems. ASIC1a is one of the most intensively studied isoforms due to its importance and wide representation in organisms, but it is still largely unexplored as a target for therapy. In this study, we demonstrated response of the ASIC1a to acidification in the presence of the daurisoline (DAU) ligand. DAU alone did not activate the channel, but in combination with protons, it produced the second peak component of the ASIC1a current. This second peak differs from the sustained component (which is induced by RF-amide peptides), as the second (DAU-induced) peak is completely desensitized, with the same kinetics as the main peak. The co-application of DAU and mambalgin-2 indicated that their binding sites do not overlap. Additionally, we found an asymmetry in the pH activation curve of the channel, which was well-described by a mathematical model based on the multiplied probabilities of protons binding with a pool of high-cooperative sites and a single proton binding with a non-cooperative site. In this model, DAU targeted the pool of high-cooperative sites and, when applied with protons, acted as an inhibitor of ASIC1a activation. Moreover, DAU's occupation of the same binding site most probably reverses the channel from steady-state desensitization in the pH 6.9-7.3 range. DAU features disclose new opportunities in studies of ASIC structure and function.

With rapid progress in medicine, a thorough understanding of pharmacology remains crucial. Currently, lecturers are implementing competency-based learning objectives in medical curricula around the world. Advancing teaching modalities need to be integrated into pharmacology courses in a reasonable way. At Ludwig-Maximilians-Universität (LMU) Munich Medical Faculty, a systematic evidence-based approach was used to modernize pharmacology classes. The needs assessment was conducted by final-year students. It included focus groups and a large-scale online survey, which was distributed among all medical students at LMU, with 1018 students participating (response rate 20%). Survey results showed that most of the students (92%) aimed to become pharmacology-adept doctors. Also, a majority (88%) stated that their goal was to understand the material most critical to application of pharmacology concepts as well as prescribing practice. Only 38% of the students reported satisfaction with the current curriculum, and 93% supported modernization. Thus far, pharmacology teaching at LMU Munich had mainly consisted of lectures attended by 200 students. Now, students advocated for a stronger integration of clinical pharmacology teaching into clinical subjects in the last 2 years of medical school. Specifically, they called for classes with smaller groups of students including problem-based learning as well as video podcasts. These results provided the foundation for change in curriculum at the LMU medical school. In conclusion, a structured approach for curriculum development that considers students' views is feasible and can reveal their actual goals and demands. The approach has proven successful at LMU Munich and is transferrable to other universities., (© 2019, The American College of Clinical Pharmacology.)

The present study gives a detailed phenotypic and genotypic characterization of three Trueperella abortisuis strains isolated from a ten year old male Hovawart dog with an abscess of anal sac, from urine of an eight year old European shorthair cat with urolithiasis and nephrolithiasis and from a 14year old Maine Coon cat with a perianal abscess, respectively. All three strains could be identified phenotypically, by MALDI-TOF MS analysis and genotypically by sequencing the 16S rDNA and the molecular target genes gap and tuf. The present study gives a first description of T. abortisuis of this origin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Serotonin (5-hydroxy-tryptamine, 5-HT) exerted concentration-dependent positive inotropic effects or positive chronotropic effects in transgenic (TG) mice which overexpress the human 5-HT 4a receptor in the heart but not in littermate wild-type (WT) mice. These positive inotropic effects and positive chronotropic effects are thought to be mediated by cyclic adenosine 3',5'-monophosphate (cAMP) in TG cardiomyocytes. To determine whether these effects are antagonized by endogenous phosphodiesterases (PDEs), the inotropic and chronotropic effects of 5-HT were tested in the additional presence of the PDE inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) (1 μM, a PDE2 inhibitor) or cilostamide (1 μM, a PDE3 inhibitor), rolipram (0.1 μM and 1 μM, a PDE4 inhibitor), and their combinations. For comparison, 3-isobutyl-1-methylxanthine (IBMX), an unspecific PDE inhibitor, was investigated. The use of 10 μM IBMX, the combination of rolipram (1 μM) and EHNA (1 μM), and the combination of rolipram (0.1 μM) and cilostamide (1 μM) each increased the potency of 5-HT to elevate the force of contraction in TG mice, but not the potency of 5-HT to increase the beating rate in TG mice. This indicates that PDE4 and PDE2 regulate the inotropic but not the chronotropic effects of 5-HT in TG mice. In contrast, cilostamide (1 μM) alone, EHNA (1 μM) alone, or in combination decreased the potency of 5-HT to increase force of contraction in TG mice. In summary, our present data suggest that the positive chronotropic effect of 5-HT in TG mice does not involve PDE activities, whereas the positive inotropic effect of 5-HT and the basal force in TG mice are diminished by endogenous activity of PDE4. Phosphorylation of PDE4, when PDE2 or PDE3 is inhibited, might enhance the activity of PDE4.

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system. AD is characterized by progressive impairments of memory as well as other cognitive functions and an increasing loss of autonomy in everyday life. This review article provides an overview of the current state-of-the-art (symptomatic) pharmacological treatment of Alzheimer's disease, specifics in the context of concomitant neuropsychiatric symptoms in multimorbid patients, and drugs currently under development that have a potentially causal (disease modifying) effect are also mentioned.

With the development of advanced focusing optics for x-rays, we can now use x-ray beams with spot sizes in the micro- or nanometer range to scan cells and large areas of tissues and continuously record the diffraction signals. From this data, x-ray scattering maps or so-called x-ray darkfield images are computed showing how different types of cells or regions of tissues differ in their diffraction intensity. At the same time a diffraction pattern is available for each scan point which encodes the local nanostructure, averaged over many contributing constituents illuminated by the beam. In this work we have exploited these new capabilities of scanning x-ray diffraction to investigate cardiac muscle cells as well as cardiac tissue. We give examples of how cardiac cells, especially living, cultured cells, can be prepared to be compatible with the instrumentation constraints of nano- or micro-diffraction instruments. Furthermore, we show how the developmental stage, ranging from neonatal to adult cells, as well as the final preparation state of the cardiomyocytes influences the recorded scattering signal and how these diffraction signals compare to the structure of a fully developed cardiac muscle., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Clostridium difficile induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence attributed to different factors. We compared cellular intoxication by TcdB NAP1 with that by the reference strain VPI 10463 (TcdB VPI ). In a mouse ligated intestinal loop model, TcdB NAP1 induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdB VPI . Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdB VPI and TcdB NAP1 might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdB NAP1v ) unable to glucosylate RhoA but with the same receptor-binding domains as TcdB NAP1 . Cells were preincubated with TcdB NAP1v to block cellular receptors, prior to intoxication with either TcdB VPI or TcdB NAP1 . Preincubation with TcdB NAP1v blocked RhoA glucosylation by TcdB NAP1 but not by TcdB VPI , indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdB NAP1 in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain.

In the present study, an attempt has been made to develop a new series of 1,3,7,8-tetrasubstituted xanthine based potent and selective AR ligands for the treatment of Parkinson's disease. Antagonistic interactions between dopamine and A 2A adenosine receptors serve as the basis for the development of AR antagonists as potential drug candidates for PD. All the synthesized compounds have been evaluated for their affinity toward AR subtypes using in vitro radioligand binding assays. 1,3-Dipropylxanthine 7a with a methyl substituent at N-7 position represents the most potent compound of the series and displayed highest affinity (A 2A , K i  = 0.108 µM), however incorporation of a propargyl group at 7-positon of the xanthine nucleus seems to be the most appropriate substitution to improve selectivity towards the A 2A subtype along with reasonable potency. Antiparkinsonian activity has been evaluated using perphenazine induced catatonia in rats. Most of the synthesized xanthines significantly lowered the catatonic score as compared to control and displayed antiparkinsonian effects comparable to standard drug. All the synthesized compounds were subjected to grid-based molecular docking studies to understand the key structural requirements for the development of new molecules well-endowed with intrinsic efficacy and selectivity as adenosine receptor ligands. In silico studies carried out on newly synthesized xanthines provided further support to the pharmacological results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b , Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aβ, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aβ than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres., (© 2019 Horsthemke et al.)

Lung fibrosis is characterized by excessive deposition of extracellular matrix (ECM), in particular collagens, by fibroblasts in the interstitium. Transforming growth factor-β1 (TGF-β1) alters the expression of many extracellular matrix (ECM) components produced by fibroblasts, but such changes in ECM composition as well as modulation of collagen post-translational modification (PTM) levels have not been comprehensively investigated. Here, we performed mass spectrometry (MS)-based proteomics analyses to assess changes in the ECM deposited by cultured lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients upon stimulation with transforming growth factor β1 (TGF-β1). In addition to the ECM changes commonly associated with lung fibrosis, MS-based label-free quantification revealed profound effects on enzymes involved in ECM crosslinking and turnover as well as multiple positive and negative feedback mechanisms of TGF-β1 signaling. Notably, the ECM changes observed in this in vitro model correlated significantly with ECM changes observed in patient samples. Because collagens are subject to multiple PTMs with major implications in disease, we implemented a new bioinformatic platform to analyze MS data that allows for the comprehensive mapping and site-specific quantitation of collagen PTMs in crude ECM preparations. These analyses yielded a comprehensive map of prolyl and lysyl hydroxylations as well as lysyl glycosylations for 15 collagen chains. In addition, site-specific PTM analysis revealed novel sites of prolyl-3-hydroxylation and lysyl glycosylation in type I collagen. Interestingly, the results show, for the first time, that TGF-β1 can modulate prolyl-3-hydroxylation and glycosylation in a site-specific manner. Taken together, this proof of concept study not only reveals unanticipated TGF-β1 mediated regulation of collagen PTMs and other ECM components but also lays the foundation for dissecting their key roles in health and disease. The proteomic data has been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the data set identifier MSV000082958., (© 2019 Published by Elsevier B.V.)

The mature mammalian myocardium contains composite junctions (areae compositae) that comprise proteins of adherens junctions as well as desmosomes. Mutations or deficiency of many of these proteins are linked to heart failure and/or arrhythmogenic cardiomyopathy in patients. We firstly wanted to address the question whether the expression of these proteins shows an age-dependent alteration in the atrium of the human heart. Right atrial biopsies, obtained from patients undergoing routine bypass surgery for coronary heart disease were subjected to immunohistology and/or western blotting for the plaque proteins plakoglobin (γ-catenin) and plakophilin 2. Moreover, the Z-band protein cypher 1 (Cypher/ZASP) and calcium handling proteins of the sarcoplasmic reticulum (SR) like phospholamban, SERCA and calsequestrin were analyzed. We noted expression of plakoglobin, plakophilin 2 and Cypher/ZASP in these atrial preparations on western blotting and/or immunohistochemistry. There was an increase of Cypher/ZASP expression with age. The present data extend our knowledge on the expression of anchoring proteins and SR regulatory proteins in the atrium of the human heart and indicate an age-dependent variation in protein expression. It is tempting to speculate that increased expression of Cypher/ZASP may contribute to mechanical changes in the aging human myocardium.

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A 1 receptors (A 1 Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A 1 R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A 1 R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A 1 R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A 1 R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in in vitro and ex vivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation in vitro in human neuroblastoma (SH-SY5Y) cells both A 1 R partial agonists increased cell viability. Considering the high level of expression of A 1 Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7 min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A 1 R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2019 International Society for Neurochemistry.)

A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.

Introduction: Alzheimer's disease (AD) is a neurodegenerative illness with genetic risk as an etiological factor in a subset of cases. In AD with autosomal dominant inheritance, the extracellular β-amyloid (Aβ) aggregates and intracellular neurofibrillary tangles which consist of hyperphosphorylated tau, appear to be involved in the neuronal damage; however, other forms of AD may have a polygenetic causality. Microglial cells orchestrate pathophysiological events responsible for neuronal damage in AD. They surround Aβ aggregates and the stimulation of microglial P2X7 receptors (P2X7Rs) by high local concentrations of ATP which originates from damaged CNS cells, results in degeneration of nearby neurons. Areas covered: We discuss the pathogenesis of Alzheimer's disease, the role of P2X7 receptors and their potential as therapeutic targets. We also address the fundamental hurdles in the development of new therapeutic strategies for Alzheimer's disease. Expert opinion: There are many difficulties associated with the development of efficient pharmacological strategies for AD; the lack of good animal and cellular models for this illness is a key obstacle. None of the pharmacological strategies developed so far have led to an improvement of the treatment of AD. Hence, the consideration of blood-brain barrier-permeable P2X7R antagonists as possible therapeutic agents in AD is a must.

Aims: B56α is a protein phosphatase 2A (PP2A) regulatory subunit that is highly expressed in the heart. We previously reported that cardiomyocyte B56α localizes to myofilaments under resting conditions and translocates to the cytosol in response to acute β-adrenergic receptor (β-AR) stimulation. Given the importance of reversible protein phosphorylation in modulating cardiac function during sympathetic stimulation, we hypothesized that loss of B56α in mice with targeted disruption of the gene encoding B56α (Ppp2r5a) would impact on cardiac responses to β-AR stimulation in vivo., Methods and Results: Cardiac phenotype of mice heterozygous (HET) or homozygous (HOM) for the disrupted Ppp2r5a allele and wild type (WT) littermates was characterized under basal conditions and following acute β-AR stimulation with dobutamine (DOB; 0.75 mg/kg i.p.) or sustained β-AR stimulation by 2-week infusion of isoproterenol (ISO; 30 mg/kg/day s.c.). Left ventricular (LV) wall thicknesses, chamber dimensions and function were assessed by echocardiography, and heart tissue collected for gravimetric, histological, and biochemical analyses. Western blot analysis revealed partial and complete loss of B56α protein in hearts from HET and HOM mice, respectively, and no changes in the expression of other PP2A regulatory, catalytic or scaffolding subunits. PP2A catalytic activity was reduced in hearts of both HET and HOM mice. There were no differences in the basal cardiac phenotype between genotypes. Acute DOB stimulation induced the expected inotropic response in WT and HET mice, which was attenuated in HOM mice. In contrast, DOB-induced increases in heart rate were unaffected by B56α deficiency. In WT mice, ISO infusion increased LV wall thicknesses, cardiomyocyte area and ventricular mass, without LV dilation, systolic dysfunction, collagen deposition or foetal gene expression. The hypertrophic response to ISO was blunted in mice deficient for B56α., Conclusion: These findings identify B56α as a potential regulator of cardiac structure and function during β-AR stimulation., (© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology.)

As a result of decentralized and non-harmonized documentation of poisoning cases within Germany, in comparison to other countries (e.g. the USA, Switzerland, Sweden, and the Netherlands), there is a substantial lack of harmonized national data, in particular for the early detection and risk assessment at the German Federal Institute for Risk Assessment (BfR).The establishment of a national monitoring system for poisonings in Germany is therefore an important step towards enabling faster and more efficient technical access to consolidated accumulated national poisoning data. Additionally, the monitoring would consolidate the publication of annual reports (modelled on the USA National Poison Data System reports) of the German Poison Information Centres and the BfR. Timely assessment of poisoning cases enables fast recognition and confirmation of acute as well as previously unrecognized poisoning hazards. Also relevant here is the safety of products and the associated protection, especially of children, against poisoning accidents.The continuous monitoring and reporting requirements of such a system necessitate additional and challenging work, suitable human resources and IT infrastructure and an annual budget that must be taken into consideration for overall as well as financial planning.

Increased late sodium current (late I Na ) is an important arrhythmogenic trigger in cardiac disease. It prolongs cardiac action potential and leads to an increased SR Ca 2+ leak. This study investigates the contribution of Ca 2+ /Calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA) and conversely acting protein phosphatases 1 and 2A (PP1, PP2A) to this subcellular crosstalk. Augmentation of late I Na (ATX-II) in murine cardiomyocytes led to an increase of diastolic Ca 2+ spark frequency and amplitudes of Ca 2+ transients but did not affect SR Ca 2+ load. Interestingly, inhibition of both, CaMKII and PKA, attenuated the late I Na -dependent induction of the SR Ca 2+ leak. PKA inhibition additionally reduced the amplitudes of systolic Ca 2+ transients. FRET-measurements revealed increased levels of cAMP upon late I Na augmentation, which could be prevented by simultaneous inhibition of Na + /Ca 2+ -exchanger (NCX) suggesting that PKA is activated by Ca 2+ -dependent cAMP-production. Whereas inhibition of PP2A showed no effect on late I Na -dependent alterations of Ca 2+ cycling, additional inhibition of PP1 further increased the SR Ca 2+ leak. In line with this, selective activation of PP1 yielded a strong reduction of the late I Na -induced SR Ca 2+ leak and did not affect systolic Ca 2+ release. This study indicates that phosphatase/kinase-balance is perturbed upon increased Na + influx leading to disruption of ventricular Ca 2+ cycling via CaMKII- and PKA-dependent pathways. Importantly, an activation of PP1 at RyR2 may represent a promising new toehold to counteract pathologically increased kinase activity.

Current guidelines for specialized treatment of diabetes mellitus in the elderly (>65 years old) are primarily based on epidemiologic studies and geriatric assessment of functional health. Yet, age-dependent alterations of glucose metabolism and homeostasis are highly relevant to the pathophysiology of diabetes in the elderly. In this review, we focus on age-related alterations in metabolic pathways and their relevance for the specialized diabetic care in the elderly. We review the role of increasing insulin resistance, age-related β‑cell dysfunction and incretin secretion. The clinical relevance of these effects will also be discussed in regard to the central geriatric syndrome of sarcopenia and antidiabetic drug therapy.

Administration of digitalis in heart failure (HF) increases quality of life but does not carry a prognostic benefit. Digitalis is an indirect inhibitor of the Na + /Ca 2+ exchanger (NCX), which is overexpressed in HF. We therefore used the cardiac glycoside ouabain in Ca 2+ imaging experiments and patch-clamp experiments in isolated ventricular myocytes from nonfailing transgenic NCX overexpressor mice (OE). In field-stimulated myocytes, ouabain (1-100 μm) increased the amplitude of the Ca 2+ transient in OE and wild-type (WT) similarly. Ouabain-mediated spontaneous Ca 2+ -activity was significantly more pronounced in OE compared to WT myocytes at higher concentrations (100 μm). Also, at very high concentrations (1000 μm) of ouabain, the number of cells with hypercontraction leading to cell death was higher in OE. Ouabain (10 μm) shortened the action potential duration in both genotypes. Our findings suggest that the proarrhythmic but not the inotropic effects of cardiac glycosides are enhanced by increased NCX expression. This may offer an explanation for the observed lack of prognostic benefit but increased quality of life in HF, which is accompanied by NCX upregulation., (© 2018 Société Française de Pharmacologie et de Thérapeutique.)

Heart failure and aging of the heart show many similarities regarding hemodynamic and biochemical parameters. There is evidence that heart failure in experimental animals and humans is accompanied and possibly exacerbated by increased activity of protein phosphatase (PP) 1 and/or 2A. Here, we wanted to study the age-dependent protein expression of major members of the protein phosphatase family in human hearts. Right atrial samples were obtained during bypass surgery. Patients ( n =60) were suffering from chronic coronary artery disease (CCS 2-3; New York Heart Association (NYHA) stage 1-3). Age ranged from 48 to 84 years (median 69). All patients included in the study were given β -adrenoceptor blockers. Other medications included angiotensin-converting enzyme (ACE) or angiotensin-receptor-1 (AT 1 ) inhibitors, statins, nitrates, and acetylsalicylic acid (ASS). 100  µ g of right atrial homogenates was used for western blotting. Antibodies against catalytic subunits (and their major regulatory proteins) of all presently known cardiac serine/threonine phosphatases were used for antigen detection. In detail, we studied the expression of the catalytic subunit of PP1 (PP1c); I 1 PP1 and I 2 PP1 , proteins that can inhibit the activity of PP1c; the catalytic subunit of PP2A (PP2Ac); regulatory A-subunit of PP2A (PP2A A ); regulatory B56 α -subunit of PP2A (PP2A B ); I 1 PP2A and I 2 PP2A , inhibitory subunits of PP2A; catalytic and regulatory subunits of calcineurin: PP2B A and PP2B B ; PP2C; PP5; and PP6. All data were obtained within the linear range of the assay. There was a significant decline in PP2Ac and I 2 PP2A expression in older patients, whereas all other parameters remained unchanged with age. It remains to be elucidated whether the decrease in the protein expression of I 2 PP2A might elevate cardiac PP2A activity in a detrimental way or is overcome by a reduced protein expression and thus a reduced activity of PP2Ac.

Aims: Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis., Methods and Results: Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAV-shControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1., Conclusions: Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload.

Pharmacotherapy of Sleep-Wake Disorders Abstract. Sleep is a complex behavior, coordinated by many different brain regions and neurotransmitters. These neurochemical systems can be pharmacologically influenced to modulate wakefulness and sleep. Excessive daytime sleepiness (EDS) is often treated with dopaminergic drugs, which in mild cases range from caffeine via (ar)modafinil to amphetamine derivatives. Tricyclic antidepressants and melatonin-based drugs are also used to promote alertness, but to a lesser extent. The drugs used to promote sleep include GABA-ergic drugs such as benzodiazepines and Z-hypnotics as well as histamine H1 receptor antagonists. Exogenous melatonin or a pharmacological combination of melatonin receptor agonists and 5-HT2C receptor antagonists are also used in mild cases. Selective and dual orexin (hypocretin) receptor antagonists (DORA) as well as drugs binding to specific 5-HT receptors are currently being investigated as future sleep-promoting drugs. However, pharmacological treatment is not always the primary treatment option, insomnia is treated first-line with cognitive behavioral therapy, and continuous positive airway pressure is used to treat sleep apnea.

Rationale: A higher expression/activity of type 1 serine/threonine protein phosphatase 1 (PP1) may contribute to dephosphorylation of cardiac regulatory proteins triggering the development of heart failure., Objective: Here, we tested the putatively protective effects of PP1 inhibitor-2 (I 2 ) overexpression using a heart failure model induced by chronic β-adrenergic stimulation., Methods and Results: Transgenic (TG) and wild-type (WT) mice were subjected to isoprenaline (ISO) or isotonic NaCl solution supplied via osmotic minipumps for 7 days. I 2 overexpression was associated with a depressed PP1 activity. Basal contractility was unchanged in catheterized mice and isolated cardiomyocytes between TG NaCl and WT NaCl . TG ISO mice exhibited more fibrosis and a higher expression of hypertrophy marker proteins as compared to WT ISO . After acute administration of ISO, the contractile response was accompanied by a higher sensitivity in TG ISO as compared to WT ISO . In contrast to basal contractility, the peak amplitude of [Ca] i and SR Ca load were reduced in TG NaCl as compared to WT NaCl . These effects were normalized to WT levels after chronic ISO stimulation. Cardiomyocyte relaxation and [Ca] i decay kinetics were hastened in TG ISO as compared to WT ISO , which can be explained by a higher phospholamban phosphorylation at Ser 16 . Chronic catecholamine stimulation was followed by an enhanced expression of GSK3β, whereas the phosphorylation at Ser 9 was lower in TG as compared to the corresponding WT group. This resulted in a higher I 2 phosphorylation that may reactivate PP1., Conclusion: Our findings suggest that the basal desensitization of β-adrenergic signaling and the depressed Ca handling in TG by inhibition of PP1 is restored by a GSK3β-dependent phosphorylation of I 2 ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The newly described type strains Arcanobacterium pinnipediorum DSM 28752 T and Arcanobacterium wilhelmae DSM 102162 T , initially isolated from an anal swab of a harbor seal (Sammra et al. Int J Syst Evol Microbiol 65:4539-4543, 2015) and the genital tract of a rhinoceros (Sammra et al. Int J Syst Evol Microbiol 67:2093-2097, 2017), could be further characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared (FT-IR) spectroscopy and by sequencing the genomic targets 16S-23S rDNA intergenic spacer region (ISR) and the genes rpoB, gap, and tuf. The two strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of both species remained unclear. However, the detection of specific spectra by MALDI-TOF MS and by FT-IR spectroscopy and the presented genotypic approaches might help to identify A. pinnipediorum and A. wilhelmae in the future and might elucidate the role these two species play in infections of animals.

The hyperpolarization-activated inward current, I h , plays a key role in the generation of rhythmic activities in thalamocortical (TC) relay neurons. Cyclic nucleotides, like 3',5'-cyclic adenosine monophosphate (cAMP), facilitate voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels by shifting the activation curve of I h to more positive values and thereby terminating the rhythmic burst activity. The role of 3',5'-cyclic guanosine monophosphate (cGMP) in modulation of I h is not well understood. To determine the possible role of the nitric oxide (NO)-sensitive cGMP-forming guanylyl cyclase 2 (NO-GC2) in controlling the thalamic I h , the voltage-dependency and cGMP/cAMP-sensitivity of I h was analyzed in TC neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in wild type (WT) and NO-GC2-deficit (NO-GC2 -/- ) mice. Whole cell voltage clamp recordings in brain slices revealed a more hyperpolarized half maximal activation (V 1/2 ) of I h in NO-GC2 -/- TC neurons compared to WT. Different concentrations of 8-Br-cAMP/8-Br-cGMP induced dose-dependent positive shifts of V 1/2 in both strains. Treatment of WT slices with lyase enzyme (adenylyl and guanylyl cyclases) inhibitors (SQ22536 and ODQ) resulted in further hyperpolarized V 1/2 . Under current clamp conditions NO-GC2 -/- neurons exhibited a reduction in the I h -dependent voltage sag and reduced action potential firing with hyperpolarizing and depolarizing current steps, respectively. Intrathalamic rhythmic bursting activity in brain slices and in a simplified mathematical model of the thalamic network was reduced in the absence of NO-GC2. In freely behaving NO-GC2 -/- mice, delta and theta band activity was enhanced during active wakefulness (AW) as well as rapid eye movement (REM) sleep in cortical local field potential (LFP) in comparison to WT. These findings indicate that cGMP facilitates I h activation and contributes to a tonic activity in TC neurons. On the network level basal cGMP production supports fast rhythmic activity in the cortex.

P2X7 receptors (Rs) mediate apoptosis/necrosis in neuronal and non-neuronal systems. Patch-clamp recordings from dentate gyrus (DG) granule cells in acutely prepared hippocampal slices of mice showed that incubation with 4-aminopyridine (4-AP) causes an excitability increase. This led to an enhanced sensitivity of P2X7Rs of the underlying subgranular zone neural progenitor cells (NPCs) towards dibenzoyl-ATP (Bz-ATP). The glutamatergic agonists NMDA and AMPA, as well as the purinergic agonist ATP also increased the Bz-ATP-induced current amplitudes (I BzATP ). Tetrodotoxin as well as the standard antiepileptic drugs phenytoin, valproic acid and gabapentin counteracted the effect of 4-AP, most likely by decreasing the firing rate and/or action potential duration of DG granule cells and in consequence the release of ATP/glutamate onto NPCs. Experiments with organotypic hippocampal slice cultures confirmed these results also under conditions when 4-AP was applied for longer time periods and at much lower concentrations than used in acute slices. It was concluded that pathological firing modelled by 4-AP might trigger a sensitivity increase of P2X7Rs leading to necrosis/apoptosis of NPCs with the subsequent decrease of NPC, and in consequence, granule cell number. Hence, supersensitive P2X7Rs may exert a beneficial counter-regulatory effect by reducing the chances for the evolution of chronic temporal lobe epilepsy by ectopically located granule cells., (Copyright © 2018. Published by Elsevier Ltd.)

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A 3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A 3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA 3 AR K i  = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A 1 , A 2A and A 3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Cisapride and prucalopride act as 5-HT 4 receptor agonists. As a part of our ongoing effort to study the utility of a transgenic (TG) mouse model overexpressing cardiac 5-HT 4 receptors, we assessed the extent to which we could recapitulate cisapride and prucalopride agonists. Contractile studies were performed using isolated left and right atrial preparations of TG mice showing cardiac-specific human 5-HT 4a receptor expression and those of their wild-type (WT) littermates. 5-Hydroxytryptamine (5-HT), cisapride, and prucalopride exerted concentration-dependent positive inotropic effects in the left atrial preparations of TG mice. Moreover, 5-HT induced concentration-dependent arrhythmias in the right atrial preparations of TG mice starting from 10-nM concentration. However, cisapride induced arrhythmias not only in the right atrial preparations of TG mice but also in the right atrial preparations of WT mice. For instance, 10 μM cisapride induced arrhythmias in the right atrial preparations of TG and WT mice to the same extent. Prucalopride did not exert concentration-dependent proarrhythmic effects in the isolated atrial preparations (left or right, WT or TG). Furthermore, cisapride and prucalopride increased the contractility and beating rate in vivo in TG mice, as assessed by performing echocardiography and surface electrocardiography. In summary, our results indicate that cisapride and prucalopride increase contractility and beating rate in the isolated atrial preparations of TG mice or in intact TG mice. Moreover, 5-HT induced arrhythmias in the isolated right atrial preparations of TG mice in a concentration-dependent manner. Furthermore, cisapride induced arrhythmias in the isolated right atrial preparations of both TG and WT mice. In contrast, prucalopride did not induce arrhythmias in the atrial preparations (left or right) of both WT and TG mice. We suggest that the present TG mouse model might be useful to predict at least some important cardiac effects of 5-HT 4 receptor agonists in the human heart.

It was previously shown that the estrogen-receptor negative breast cancer cell line MBA-MD-231 expresses high levels of A2B adenosine receptors as the sole adenosine receptor subtype. These receptors couple to both, stimulation of adenylyl cyclase and a Ca2+ signal. In order to establish a potential role of A2B adenosine receptors in tumor growth and development MAPK signaling was investigated in these breast cancer cells. Although it is known that A2B adenosine receptors may stimulate MAPK it was found that in MBA-MD-231 cells ERK1/2 phosphorylation is reduced upon agonist-stimulation of A2B adenosine receptors. This reduction is also triggered by forskolin, but abolished by the PKA inhibitor H89, suggesting an important role for the cAMP-PKA pathway. Likewise, a role for intracellular Ca2+ was established as the Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) abolished the reduction of ERK1/2 phosphorylation triggered by A2B stimulation. It was shown that various pathways downstream from A2B adenosine receptors resulted in a stimulation of MAPK phosphatase-1 (MKP-1) which dephosphorylates phospho ERK1/2, and thus plays a critical role in the regulation of the phosphorylation state of ERK1/2. The reduction of ERK1/2 phosphorylation mediated by A2B adenosine receptors might provide an interesting approach for adjuvant treatment leading to reduced growth of certain tumors expressing the A2B subtype., Competing Interests: The authors have declared that no competing interests exist.

The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.

About half of the cardiac serine/threonine phosphatase activity is due to the activity of protein phosphatase type 1 (PP1). The activity of PP1 can be inhibited by an endogenous protein for which the expression inhibitor-2 (I-2) has been coined. We have previously described a transgenic mouse overexpressing a truncated form of I-2. Here, we have described and initially characterized several founders that overexpress the non-truncated (i.e., full length) I-2 in the mouse heart (TG) and compared them with non-transgenic littermates (WT). The founder with the highest overexpression of I-2 displayed under basal conditions no difference in contractile parameters (heart rate, developed tension, and its first derivate) compared to WT. The relative level of PP1 inhibition was similar in mice overexpressing the non-truncated as well as the truncated form of I-2. For comparison, we overexpressed I-2 by an adenoviral system in several cell lines (myocytes from a tumor-derived cell line (H9C2), neonatal rat cardiomyocytes, smooth muscle cells from rat aorta (A7R5)). We noted gene dosage-dependent staining for I-2 protein in infected cells together with reduced PP1 activity. Finally, I-2 expression in neonatal rat cardiomyocytes led to an increase of Ca 2+ transients by about 60%. In summary, we achieved immunologically confirmed overexpression of wild-type I-2 in cardiovascular cells which was biochemically able to inhibit PP1 in the whole heart (using I-2 transgenic mice) as well as in isolated cells including cardiomyocytes (using I-2 coding virus) indirectly underscoring the importance of PP1 for cardiovascular function.

Background: Atrial fibrillation (AF) is a significant cause of morbidity and mortality with foreseeably increasing prevalence. While large animal models of the disease are well established but resource intensive, transgenic AF mouse models are not yet widely used to develop or validate novel therapeutics for AF. Hemizygous mice with a cardiomyocyte-specific overexpression of the human cAMP response element modulator (CREM) isoform IbΔC-X spontaneously develop AF on grounds of an arrhythmogenic substrate consisting of alterations in structure, conduction, and calcium handling. Objective: We investigated if homozygous expression of the CREM-IbΔC-X transgene in mice alters the time course of AF development, and if homozygous CREM-IbΔC-X transgenics could be suitable as a disease model of AF. Methods: Southern Blot, quantitative real-time PCR, and immunoblotting were used to identify and verify homozygous transgenics. Cardiac gravimetry, quantitative real-time RT-PCR, histology, survival analysis, and repeated ECG recordings allowed assessment of phenotypic development and effects of antiarrhythmic drugs. Results: Homozygous animals could be identified by Southern blot and quantitative PCR, showing a strong trend to increased transgenic protein expression. In homozygous animals, atrial hypertrophy appeared earlier and more pronounced than in hemizygous animals, going along with an earlier onset of spontaneous AF, while no increased early mortality was observed. Application of a rate-controlling drug (esmolol) led to the expected result of a decreased heart rate. Application of a rhythm-controlling drug (flecainide) showed effects on heart rate variability, but did not lead to a definitive conversion to sinus rhythm. Conclusion: We suggest homozygous CREM-IbΔC-X overexpressing mice as a reliable model of early onset, rapidly progressive AF.

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-β42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.

The synthesis of 9-alkyl substituted adenine derivatives presenting aromatic groups and cycloalkyl rings in 8- and N 6 -position, respectively, is reported. The compounds were tested with radioligand binding studies showing, in some cases, a low nanomolar A 1 adenosine receptor affinity and a very good selectivity versus the other adenosine receptor subtypes. Functional assays at human adenosine receptors and at a mouse ileum tissue preparation clearly demonstrate the antagonist profile of these molecules, with inhibitory potency at nanomolar level. A molecular modeling study, consisting in docking analysis at the recently reported A 1 adenosine receptor crystal structure, was performed for the interpretation of the obtained pharmacological results. The N 6 -cyclopentyl-9-methyl-8-phenyladenine (17), resulting the most active derivative of the series (K i  = 2.8 nM and IC 50  = 14 nM), was also very efficacious in counteracting the effect of the agonist CCPA on mouse ileum contractility. This new compound represents a tool for the development of new agents for the treatment of intestinal diseases as constipation and postoperative ileus., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Two subclasses of acid-sensing ion channels (ASIC3) and of ATP-sensitive P2X receptors (P2X3Rs) show a partially overlapping expression in sensory neurons. Here we report that both recombinant and native receptors interact with each other in multiple ways. Current measurements with the patch-clamp technique prove that ASIC3 stimulation strongly inhibits the P2X3R current partly by a Ca 2+ -dependent mechanism. The proton-binding site is critical for this effect and the two receptor channels appear to switch their ionic permeabilities during activation. Co-immunoprecipation proves the close association of the two protein structures. BN-PAGE and SDS-PAGE analysis is also best reconciled with the view that ASIC3 and P2X3Rs form a multiprotein structure. Finally, in vivo measurements in rats reveal the summation of pH and purinergically induced pain. In conclusion, the receptor subunits do not appear to form a heteromeric channel, but tightly associate with each other to form a protein complex, mediating unidirectional inhibition.

l-homoarginine (l-hArg) is an endogenous non-proteinogenic amino acid. Low l-hArg concentrations are associated with increased all-cause mortality, fatal strokes, and worse outcome after stroke. On the other hand, oral supplementation with l-hArg in mice improved neurological deficits and preserved cardiac function in experimental models of stroke and heart failure, respectively. Recently, oral supplementation with 125 mg daily l-hArg capsules in healthy volunteers demonstrated increased l-hArg plasma levels. Therefore, oral l-hArg supplementation could represent a potential treatment for patients with cerebrovascular disease. In addition to vascular physiology, animal studies have suggested that l-hArg might play a role in synapse function, neurotransmitter metabolism and cognitive training. However the direct influence of l-hArg on cognitive function has not been studied so far. In this study, cognitive performance in healthy humans was analyzed concerning memory, learning, and attention following supplementation with placebo or l-hArg for 4 weeks. Our results did not reveal any effects on cognition, neither impairment nor improvement, upon l-hArg supplementation. Therefore, potential l-hArg treatment is not expected to cause any acute neurocognitive or behavioral side effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A small-molecule combinatorial library of 24 compounds with 2-aminoimidazole and 2-aminoimidazolyl-thiazole derivatives was synthesized using a 2-chloro trityl resin. The generated compound library was tested against all the human adenosine receptors subtypes. The 2-aminoimidazole derivatives ( 6a-6l ) showed weak to moderate affinity towards the human adenosine receptors. Further modification to 2-aminoimidazolyl-thiazole derivatives ( 12a-12l ) resulted in an improvement of affinity at adenosine A 1 , A 2A and A 3 receptor subtypes. Compound 12b was the most potent and selective non-xanthine human adenosine A 3 receptor antagonist of this series. A receptor-based modeling study was performed to explore the possible binding mode of these novel 2-aminoimidazole and 2-aminoimidazolyl-thiazole derivatives into human adenosine A 1 , A 2A and A 3 receptor subtypes.

In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002 T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Thyroid hormone (TH) is crucial for brain development and function. This becomes most evident in untreated congenital hypothyroidism, leading to irreversible mental retardation. Likewise, maternal hypothyroxinaemia, a lack of TH during pregnancy, is associated with neurological dysfunction in the offspring, such as autism and reduced intellectual capacity. In the brain, TH acts mainly through TH receptor α1 (TRα1). Consequently, mice heterozygous for a dominant-negative mutation in TRα1 display profound neuroanatomical abnormalities including deranged development of parvalbumin neurones. However, the exact timing and orchestration of TH signalling during parvalbumin neurone development remains elusive. In the present study, we dissect the development of parvalbumin neurones in the anterior hypothalamic area (AHA) in male mice using different mouse models with impaired pre- and postnatal TH signalling in combination with bromodeoxyuridine birth dating and immunohistochemistry. Our data reveal that hypothalamic parvalbumin neurones are born at embryonic day 12 and are first detected in the AHA at postnatal day 8, reaching their full population number at P13. Interestingly, they do not require TH postnatally because their development is not impaired in mice with impaired TH signalling after birth. By contrast, however, these neurones crucially depend on TH through TRα1 signalling in the second half of pregnancy, when the hormone is almost exclusively provided by the mother. For the first time, our findings directly link a maternal hormone to a neuroanatomical substrate in the foetal brain, and underline the importance of proper TH signalling during pregnancy for offspring mental health. Given the role of hypothalamic parvalbumin neurones in the central control of blood pressure, the present study advocates the inclusion of cardiovascular parameters in the current discussion on possible TH substitution in maternal hypothyroxinaemia., (© 2018 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

Diacylglycerol-sensitive transient receptor potential (TRP) channels play crucial roles in a wide variety of biological processes and systems, but their activation mechanism is not well understood. We describe an optical toolkit by which activation and deactivation of these ion channels can be controlled with unprecedented speed and precision through light stimuli. We show that the photoswitchable diacylglycerols PhoDAG-1 and PhoDAG-3 enable rapid photoactivation of two DAG-sensitive TRP channels, Trpc2 and TRPC6, upon stimulation with UV-A light, whereas exposure to blue light terminates channel activation. PhoDAG photoconversion can be applied in heterologous expression systems, in native cells, and even in mammalian tissue slices. Combined laser scanning-controlled photoswitching and Ca 2+ imaging enables both large-scale mapping of TRP channel-mediated neuronal activation and localized mapping in small cellular compartments. Light-switchable PhoDAGs provide an important advance to explore the pathophysiological relevance of DAG-sensitive TRP channels in the maintenance of body homeostasis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Adenosine can be produced in the heart and acts on cardiac adenosine receptors. One of these receptors is the A 2A -adenosine receptor (A 2A -AR). Methods and Results: To better understand its role in cardiac function, we generated and characterized mice (A 2A -TG) which overexpress the human A 2A -AR in cardiomyocytes. In isolated atrial preparations from A 2A -TG but not from WT, CGS 21680, an A 2A -AR agonist, exerted positive inotropic and chronotropic effects. In ventricular preparations from A 2A -TG but not WT, CGS 21680 increased the cAMP content and the phosphorylation state of phospholamban and of the inhibitory subunit of troponin in A 2A -TG but not WT. Protein expression of phospholamban, SERCA, triadin, and junctin was unchanged in A 2A -TG compared to WT. Protein expression of the α-subunit of the stimulatory G-protein was lower in A 2A -TG than in WT but expression of the α-subunit of the inhibitory G-protein was higher in A 2A -TG than in WT. While basal hemodynamic parameters like left intraventricular pressure and echocardiographic parameters like the systolic diameter of the interventricular septum were higher in A 2A -TG than in WT, after β-adrenergic stimulation these differences disappeared. Interestingly, A 2A -TG hearts sustained global ischemia better than WT. Conclusion: We have successfully generated transgenic mice with cardiospecific overexpression of a functional A 2A -AR. This receptor is able to increase cardiac function per se and after receptor stimulation. It is speculated that this receptor may be useful to sustain contractility in failing human hearts and upon ischemia and reperfusion.

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the β subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)