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BackgroundChronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.Methods and resultsWe applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a β-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as β-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing β-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

We have recently identified a pool of intracellular β1 adrenergic receptors (β1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular β1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized β1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local β1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-β1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-β1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Beta-mannans are insoluble plant polysaccharides with beta-1,4-linked mannose as the backbone. We used three forms of this polysaccharide, namely, pure mannan, glucomannan, and galactomannan, to enrich haloarchaea, which have the ability to utilize mannans for growth. Four mannan-utilizing strains obtained in pure cultures were closely related to each other on the level of the same species. Furthermore, another strain selected from the same habitats with a soluble beta-1,4-glucan (xyloglucan) was also able to grow with mannan. The phylogenomic analysis placed the isolates into a separate lineage of the new genus level within the family Natrialbaceae of the class Halobacteria. The strains are moderate alkaliphiles, extremely halophilic, and aerobic saccharolytics. In addition to the three beta-mannan forms, they can also grow with cellulose, xylan, and xyloglucan. Functional genome analysis of two representative strains demonstrated the presence of several genes coding for extracellular endo- beta-1,4-mannanase from the GH5_7 and 5_8 subfamilies and the GH26 family of glycosyl hydrolases. Furthermore, a large spectrum of genes encoding other glycoside hydrolases that were potentially involved in the hydrolysis of cellulose and xylan were also identified in the genomes. A comparative genomics analysis also showed the presence of similar endo-beta-1,4-mannanase homologs in the cellulotrophic genera Natronobiforma and Halococcoides. Based on the unique physiological properties and the results of phylogenomic analysis, the novel mannan-utilizing halolarchaea are proposed to be classified into a new genus and species Natronoglomus mannanivorans gen. nov., sp. nov. with the type strain AArc-m2/3/4 (=JCM 34861=UQM 41565). [ABSTRACT FROM AUTHOR]

Abstract Aims Agonistic antibodies against neurohumoral receptors can induce cardio‐noxious effects by altering the baseline receptor activity. To estimate the prevalence of autoantibodies directed against the beta‐1 receptor (b1‐AAB) in patients admitted to the hospital for acute heart failure (HF) at (i) baseline and (ii) after 6 months of follow‐up (F6) and (iii) after another 12 months of follow‐up (i.e. 18 months after index hospitalization), to estimate their prognostic impact on clinical outcome (death or first hospitalization for HF). Methods and results In 47 patients, b1‐AAB were serially determined in serum samples collected at index hospitalization and at 6 months of follow‐up (F6) with a flow cytometry‐based assay: median age 71 years (quartiles 60, 80), 23 (49%) women, 24 (51%) HF with preserved ejection fraction. Beta1‐AAB were detected in three subjects at index hospitalization (6%), and in eight subjects at F6 (17%). There were no differences apparent between patients with and without b1‐AAB at F6 with regard to age, sex, type, duration, or main cause of HF. During the 12 month period following F6 (i.e. up to month 18), eight events occurred. Event‐free survival was associated with prevalence of b1‐AAB at F6. Compared with patients without b1‐AAB at F6, age‐adjusted Cox regression indicated a higher event risk in patients harbouring b1‐AAB, with a hazard ratio of 8.96 (95% confidence interval 1.81–44.50, P = 0.007). Conclusions Our results suggest a possible adverse prognostic relevance of b1‐AAB in patients with acute HF, but this observation needs to be confirmed in larger patient collectives.

Aims: Agonistic antibodies against neurohumoral receptors can induce cardio‐noxious effects by altering the baseline receptor activity. To estimate the prevalence of autoantibodies directed against the beta‐1 receptor (b1‐AAB) in patients admitted to the hospital for acute heart failure (HF) at (i) baseline and (ii) after 6 months of follow‐up (F6) and (iii) after another 12 months of follow‐up (i.e. 18 months after index hospitalization), to estimate their prognostic impact on clinical outcome (death or first hospitalization for HF). Methods and results: In 47 patients, b1‐AAB were serially determined in serum samples collected at index hospitalization and at 6 months of follow‐up (F6) with a flow cytometry‐based assay: median age 71 years (quartiles 60, 80), 23 (49%) women, 24 (51%) HF with preserved ejection fraction. Beta1‐AAB were detected in three subjects at index hospitalization (6%), and in eight subjects at F6 (17%). There were no differences apparent between patients with and without b1‐AAB at F6 with regard to age, sex, type, duration, or main cause of HF. During the 12 month period following F6 (i.e. up to month 18), eight events occurred. Event‐free survival was associated with prevalence of b1‐AAB at F6. Compared with patients without b1‐AAB at F6, age‐adjusted Cox regression indicated a higher event risk in patients harbouring b1‐AAB, with a hazard ratio of 8.96 (95% confidence interval 1.81–44.50, P = 0.007). Conclusions: Our results suggest a possible adverse prognostic relevance of b1‐AAB in patients with acute HF, but this observation needs to be confirmed in larger patient collectives. [ABSTRACT FROM AUTHOR]

Aimsβ-blockers are widely used in therapy for heart failure and hypertension. β-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by β-blockers in animal hearts.Methods and resultsWild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure β-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes β1 adrenergic receptor (β1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote β1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust β1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced β1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts.ConclusionThese observations present a novel β1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

Dense innervation of the heart by the sympathetic nervous system (SNS) allows cardiac output to respond appropriately to the needs of the body under varying conditions, but occasionally the abrupt onset of SNS activity can trigger cardiac arrhythmias. Sympathetic activity leads to the release of norepinephrine (NE) onto cardiomyocytes, activating β1-adrenergic receptors (β1-ARs) and leading to the production of the second messenger cyclic AMP (cAMP). Upon sudden activation of β1-ARs in experiments, intracellular cAMP can transiently rise to a high concentration before converging to a steady state level. Although changes to cellular cAMP concentration are important in modulating the overall cardiovascular response to sympathetic tone, the underlying mechanisms of the cAMP transients and the parameters that control their magnitude are unclear. We reduce a detailed computational model of the β1-adrenergic signaling cascade to a system of two differential equations by eliminating extraneous variables and applying quasi-steady state approximation. The structure of the reduced model reveals that the large cAMP transients associated with abrupt β1-AR activation are generated by the interplay of production/degradation of cAMP and desensitization/resensitization of β1-ARs. The reduced model is used to predict how the dynamics of intracellular cAMP depend on the concentrations of norepinephrine (NE), phosphodiesterases 3 and 4 (PDE3,4), G-protein coupled receptor kinase 2 (GRK2), and β1-AR, in healthy conditions and a simple model of early stages of heart failure. The key findings of the study are as follows: 1) Applying a reduced model of the dynamics of cardiac sympathetic signaling we show that the concentrations of two variables, cAMP and non-desensitized β1-AR, capture the overall dynamics of sympathetic signaling; 2) The key factors influencing cAMP production are AC activity and PDE3,4 activity, while those that directly impact β1-AR phosphorylation are GRK2 and PKA1. Thus, disease states that affect sympathetic control of the heart can be thoroughly assessed by studying AC activity, PDE3,4, GRK2 and PKA activity, as these factors directly impact cAMP production/degradation and β1-AR (de) phosphorylation and are therefore predicted to comprise the most effective pharmaceutical targets in diseases affecting cardiac β1-adrenergic signaling.

Lactose is the primary carbohydrate in the milk of most mammals and is unique in that it is only synthesized by epithelial cells in the mammary glands. Lactose is also essential for the development and nutrition of infants. Across species, the concentration of lactose in milk holds a strong positive correlation with overall milk volume. Additionally, there is a range of examples where the onset of lactose synthesis as well as the content of lactose in milk varies between species and throughout a lactation. Despite this diversity, the precursors, genes, proteins and ions that regulate lactose synthesis have not received the depth of study they likely deserve relative to the significance of this simple and abundant molecule. Through this review, our objective is to highlight the requirements for lactose synthesis at the biochemical, cellular and temporal levels through a comparative approach. This overview also serves as the prelude to a companion review describing the dietary, hormonal, molecular, and genetic factors that regulate lactose synthesis.

Beta adrenergic receptors (βARs) mediate physiologic responses to the catecholamines epinephrine and norepinephrine released by the sympathetic nervous system. While the hormone epinephrine binds β1AR and β2AR with similar affinity, the smaller neurotransmitter norepinephrine is approximately tenfold selective for the β1AR. To understand the structural basis for this physiologically important selectivity, we solved the crystal structures of the human β1AR bound to an antagonist carazolol and different agonists including norepinephrine, epinephrine and BI-167107. Structural comparison revealed that the catecholamine-binding pockets are identical between β1AR and β2AR, but the extracellular vestibules have different shapes and electrostatic properties. Metadynamics simulations and mutagenesis studies revealed that these differences influence the path norepinephrine takes to the orthosteric pocket and contribute to the different association rates and thus different affinities.

Rationaleβ1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated.ObjectiveTest localization and function of intracellular β1AR on cardiac contractility.Methods and resultsMembrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.ConclusionsFunctional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3.

BackgroundCardiac gene expression and arrhythmia occurrence have time-of-day variation; however, daily changes in cardiac electrophysiology, arrhythmia susceptibility, and Ca2+ handling have not been characterized. Furthermore, how these patterns change with age is unknown.MethodsHearts were isolated during the light (zeitgeber time [ZT] 4 and ZT9) and dark cycle (ZT14 and ZT21) from adult (12-18 weeks) male mice. Hearts from aged (18-20 months) male mice were isolated at ZT4 and ZT14. All hearts were Langendorff-perfused for optical mapping with voltage- and Ca2+-sensitive dyes (n=4-7/group). Cardiac gene and protein expression were assessed with real-time polymerase chain reaction (n=4-6/group) and Western blot (n=3-4/group).ResultsAdult hearts had the shortest action potential duration (APD) and Ca2+ transient duration (CaTD) at ZT14 (APD80: ZT4: 45.4±4.1 ms; ZT9: 45.1±8.6 ms; ZT14: 34.7±4.2 ms; ZT21: 49.2±7.6 ms, P

RationaleCardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.ObjectiveWe aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.Methods and resultsThe integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.ConclusionsThese data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires sympathetic innervation of the spleen, the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion (SMG) prevented macrophage activation and LPS-induced TNFα production in the spleen and MLN, but not in the inguinal LN. Site selective stimulation of the SMG induced the release of norepinephrine, resulting in β2AR dependent acetylcholine release in the MLN and spleen. VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen was significantly reduced using selective optogenetic blockade applied at the SMG. Additionally, this optogenetic blockade restored LPS-induced TNFα production, despite VNS. These studies identify the superior mesenteric ganglion as a critical node in a neuro-immune circuit that can inhibit immune function in the MLN and the spleen.

Sleep is crucial for our survival, and many diseases are linked to long-term poor sleep quality. Before we can use sleep to enhance our health and performance and alleviate diseases associated with poor sleep, a greater understanding of sleep regulation is necessary. We have identified a mutation in the β1-adrenergic receptor gene in humans who require fewer hours of sleep than most. In vitro, this mutation leads to decreased protein stability and dampened signaling in response to agonist treatment. In vivo, the mice carrying the same mutation demonstrated short sleep behavior. We found that this receptor is highly expressed in the dorsal pons and that these ADRB1+ neurons are active during rapid eye movement (REM) sleep and wakefulness. Activating these neurons can lead to wakefulness, and the activity of these neurons is affected by the mutation. These results highlight the important role of β1-adrenergic receptors in sleep/wake regulation.

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of β1 adrenergic receptors (βARs) on cardiac myocytes. Blocking these receptors is part of the basis for β-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including βARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi βARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface βAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident β1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious β-blocker therapies.

Increased β-adrenergic receptor (β-AR)-mediated activation of adenylyl cyclase (AC) in rat liver during aging has been linked to age-related increases in hepatic glucose output and hepatosteatosis. In this study, we investigated the expression of β-ARs, individual receptor subtypes, and G protein-coupled receptor (GPCR) regulatory proteins in livers from aging rats. Radioligand-binding studies demonstrated that β-AR density increased by greater than threefold in hepatocyte membranes from senescent (24-mo-old) compared with young adult (7-mo-old) rats and that this phenomenon was blocked by food restriction, which is known to retard aging processes in rodents. Competition-binding studies revealed a mixed population of β1- and β2-AR subtypes in liver membranes over the adult life span, with a trend for greater β2-AR density with age. Expression of both β-AR subtype mRNAs in rat liver increased with age, whereas β2- but not β1-AR protein levels declined in livers of old animals. Immunoreactive β2- but not β1-ARs were preferentially distributed in pericentral hepatic regions. Levels of GRK2/3 and β-arrestin 2 proteins, which are involved in downregulation of agonist-activated GPCRs, including β-ARs, increased during aging. Insofar as sympathetic tone increases with age, our findings suggest that, despite enhanced agonist-mediated downregulation of hepatic β-ARs preferentially affecting the β2-AR subtype, increased generation of both receptor subtypes during aging augments the pool of plasma membrane-bound β-ARs coupled to AC in hepatocytes. This study thus identifies one or both β-AR subtypes as possible therapeutic targets involved in aberrant hepatic processes of glucose and lipid metabolism during aging.

Invasive aspergillosis (IA) is a life-threatening infection. Early and specific diagnosis is pivotal to ensure adequate therapy. Antigen testing from blood is a widespread and convenient diagnostic approach. Various tests for the detection of Aspergillus antigen as well as for the panfungal antigen β-1,3-D-glucan (BDG) are available, for which comprehensive comparisons are still lacking. Blood samples of 82 proven/probable (11/71) IA patients and 52 controls were tested using two enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad and Euroimmun), one chemiluminescent immunoassay (CLIA) (Vircell), one BDG assay (Fujifilm Wako), and two point of care (PoC) assays (Immy sōna and OLM). PoC assays were evaluated visually and used automated read out systems. Of the 82 IA patients, 37 had received solid organ transplantation (SOT) and 25 hematopoietic stem cell transplant (HSCT). Sensitivities and specificities for the eight test systems ranged from 27% to 71% and from 64% to 100%. Estimating a 10% prevalence of IA, test performance would have resulted in positive and negative predictive values of 14%-100% and 91%-95%. Areas under the curve (AUCs) for all tests except GM were below 0.7. When the cut-off values for quantitative tests were normalized to a specificity close to 95%, sensitivities ranged from 14% to 40%. The use of automated read out systems for the PoC assays had a significant impact. Combining different tests did not result in better test strategies. Sensitivity of Aspergillus antigen testing from single serum samples is low. Due to specificity issues, the majority of tests is not suited for screening purposes. The different assays can meet different needs in different diagnostic settings.

β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the β1AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the β1AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies β1AR N-terminal O-glycosylation at Ser37/Ser41 as a mechanism that prevents β1AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated β1ARs and N-terminally truncated glycosylation-defective β1ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes β1ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of β1AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The β2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human β1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that β-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.

AimsCardiac β-adrenergic receptor (βAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists.Methods and resultsRat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function.ConclusionsThese data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.

β-adrenoceptor antagonists are commonly used in ischaemic heart disease (IHD) patients, yet may impair signalling and efficacy of 'cardioprotective' interventions. We assessed effects of chronic β1-adrenoceptor antagonism on myocardial resistance to ischaemia-reperfusion (IR) injury and the ability of cardioprotective interventions [classic ischaemic preconditioning (IPC); novel sustained ligand-activated preconditioning (SLP)] to reduce IR injury in murine hearts. Young male C57Bl/6 mice were untreated or received atenolol (0.5g/l in drinking water) for 4 weeks. Subsequently, two cardioprotective stimuli were evaluated: morphine pellets implanted (to induce SLP, controls received placebo) 5 days prior to Langendorff heart perfusion, and IPC in perfused hearts (3×1.5min ischaemia/2min reperfusion). Atenolol significantly reduced in vivo heart rate. Untreated control hearts exhibited substantial left ventricular dysfunction (~50% pressure development recovery, ~20mmHg diastolic pressure rise) with significant release of lactate dehydrogenase (LDH, tissue injury indicator) after 25min ischaemia/45min reperfusion. Contractile dysfunction and elevated LDH were reduced >50% with IPC and SLP. While atenolol treatment did not modify baseline contractile function, post-ischaemic function was significantly depressed compared to untreated hearts. Atenolol pre-treatment abolished beneficial effects of IPC, whereas SLP protection was preserved. These data indicate that chronic β1-adrenoceptor blockade can exert negative effects on functional IR tolerance and negate conventional IPC (implicating β1-adrenoceptors in IR injury and IPC signalling). However, novel morphine-induced SLP is resistant to inhibition by β1-adrenoceptor antagonism.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick's specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and β-1,4 galactosyltransferases (β-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars—GlcNAc, Glc, and Xyl—in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in β-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and β-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS. [ABSTRACT FROM AUTHOR]

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose-α-1, 3-galactose (α-gal) injected into humans from the lone-star tick (Amblyomma americanum) bite. Indeed, α-gal is discovered in salivary glands of lone-star tick; however, the tick’s specific intrinsic factors involved in endogenous α-gal production and presentation to host during hematophagy are poorly understood. This study aimed to investigate the functional role of two tick enzymes, α-D-galactosidase (ADGal) and β-1,4 galactosyltransferases (β-1,4GalT), in endogenous α-gal production, carbohydrate metabolism, and N-glycan profile in lone-star tick. The ADGal enzyme cleaves terminal α-galactose moieties from glycoproteins and glycolipids, whereas β-1,4GalT transfers α-galactose to a β1,4 terminal linkage acceptor sugars—GlcNAc, Glc, and Xyl—in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and β-1,4GalT in Am. americanum to examine their function in α-gal metabolism in tick and AGS onset. Silencing of ADGal led to the significant downregulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in α-gal levels in silenced tissues. However, there was no significant difference in the level of α-gal in β-1,4GalT-silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal-silenced salivary glands. These results provide an insight into the roles of ADGal and β-1,4GalT in α-gal production and presentation in ticks and the probable involvement in the onset of AGS.

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.

The counter-regulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well studied, whereas the details of insulin regulation of β adrenergic receptor (βAR) signaling pathway in heart remain unknown. Here, we characterize a novel signaling pathway of insulin receptor (IR) to G protein-coupled receptor kinase 2 (GRK2) in the heart. Insulin stimulates recruitment of GRK2 to β2AR, which induces β2AR phosphorylation at the GRK sites of serine 355/356 and subsequently β2AR internalization. Insulin thereby suppresses βAR-induced cAMP-PKA activities and contractile response in neonatal and adult mouse cardiomyocytes. Deletion of insulin receptor substrate 2 (IRS2) disrupts the complex of IR and GRK2, which attenuates insulin-mediated β2AR phosphorylation at the GRK sites and β2AR internalization, and the counter-regulation effects of insulin on βAR signaling. These data indicate the requirements of IRS2 and GRK2 for insulin to stimulate counter-regulation of βAR via β2AR phosphorylation and internalization in cardiomyocytes.

The autonomic nervous system (ANS) conveys neuronal input from the brain to the stomach. We investigated mechanisms through which urocortin 1 (UCN1) injected intracerebroventricularly (ICV, 300 pmol/rat) inhibits circulating ghrelin in rats. This was achieved by assessing (1) the induction of c-fos gene expression as a marker of neuronal activation in specific hypothalamic and caudal brainstem regulating ANS; (2) the influence of vagotomy and pharmacological blockade of central and peripheral α- and β-adrenergic receptor (AR) on ICV UCN1-induced reduction of plasma ghrelin levels (determined by ELISA); and (3) the relevance of this pathway in the feeding response to a fast in rats. UCN1 increased c-fos mRNA expression in key brain sites influencing sympathetic activity namely the hypothalamic paraventricular and ventromedial nuclei, locus coeruleus, nucleus of the solitary tract, and rostral ventrolateral medulla, by 16-, 29-, 6-, 37-, and 13-fold, respectively. In contrast, the dorsal motor nucleus of the vagus had little c-fos mRNA expression and ICV UCN1 induced a similar reduction in acylated ghrelin in the sham-operated (31%) and vagotomized (41%) rats. An intraperitoneal (IP) injection of either a non-selective α- or selective α2-AR antagonist reduced, while a selective α2-AR agonist enhanced ICV UCN1-induced suppression of plasma acylated ghrelin levels. In addition, IP injection of a non-selective β- or selective β1-AR agonist blocked, and selective β1-AR antagonist augmented, the ghrelin response to ICV UCN1. The IP injections of a selective α1- or non-selective β or β2-AR antagonists, or any of the pretreatments given ICV had no effect. ICV UCN1 reduced the 2-h food intake in response to a fast by 80%, and this effect was partially prevented by a selective α2-AR antagonist. These data suggest that ICV UCN1 reduces plasma ghrelin mainly through the brain sympathetic component of the ANS and peripheral AR specifically α2-AR activation and inactivation of β1-AR. The α2-AR pathway contributes to the associated reduction in food intake.

Use of curldlan, an insoluble β-1,3-glucan, as an enrichment substrate under aerobic conditions resulted in the selection from hypersaline soda lakes of a single natronarchaeon, strain AArc-curdl1. This organism is an obligately aerobic saccharolytic, possessing a poorly explored (in Archaea) potential to utilize beta-1–3 glucans, being only a second example of a haloarchaeon with this ability known in pure culture. The main phenotypic property of the isolate is the ability to grow with insoluble β-1,3-backboned glucans, i.e. curdlan and pachyman. Furthermore, the strain utilized starch family α-glucans, beta-fructan inulin and a limited spectrum of sugars. The major ether-bound membrane polar phospholipids included PGP-Me and PG. The glyco- and sulfolipids were absent. The major respiratory menaquinone is MK-8:8. According to phylogenomic analysis, AArc-curdl1 represents a separate species in the recently described genus Natronosalvus within the family Natrialbaceae. The closest related species is Natronosalvus amylolyticus (ANI, AAI and DDH values of 90.2, 91.6 and 44 %, respectively). On the basis of its unique physiological properties and phylogenomic distance, strain AArc-curdl1T is classified as a novel species Natronosalvus hydrolyticus sp. nov. (=JCM 34865 = UQM 41566). [ABSTRACT FROM AUTHOR]

Olivibacter sitiensis Ntougias et al. 2007 is a member of the family Sphingobacteriaceae, phylum Bacteroidetes. Members of the genus Olivibacter are phylogenetically diverse and of significant interest. They occur in diverse habitats, such as rhizosphere and contaminated soils, viscous wastes, composts, biofilter clean-up facilities on contaminated sites and cave environments, and they are involved in the degradation of complex and toxic compounds. Here we describe the features of O. sitiensis AW-6(T), together with the permanent-draft genome sequence and annotation. The organism was sequenced under the Genomic Encyclopedia for Bacteria and Archaea (GEBA) project at the DOE Joint Genome Institute and is the first genome sequence of a species within the genus Olivibacter. The genome is 5,053,571 bp long and is comprised of 110 scaffolds with an average GC content of 44.61%. Of the 4,565 genes predicted, 4,501 were protein-coding genes and 64 were RNA genes. Most protein-coding genes (68.52%) were assigned to a putative function. The identification of 2-keto-4-pentenoate hydratase/2-oxohepta-3-ene-1,7-dioic acid hydratase-coding genes indicates involvement of this organism in the catechol catabolic pathway. In addition, genes encoding for β-1,4-xylanases and β-1,4-xylosidases reveal the xylanolytic action of O. sitiensis.

Insulin and adrenergic stimulation are two divergent regulatory systems that may interact under certain pathophysiological circumstances. Here, we characterized a complex consisting of insulin receptor (IR) and β2-adrenergic receptor (β2AR) in the heart. The IR/β2AR complex undergoes dynamic dissociation under diverse conditions such as Langendorff perfusions of hearts with insulin or after euglycemic-hyperinsulinemic clamps in vivo. Activation of IR with insulin induces protein kinase A (PKA) and G-protein receptor kinase 2 (GRK2) phosphorylation of the β2AR, which promotes β2AR coupling to the inhibitory G-protein, Gi. The insulin-induced phosphorylation of β2AR is dependent on IRS1 and IRS2. After insulin pretreatment, the activated β2AR-Gi signaling effectively attenuates cAMP/PKA activity after β-adrenergic stimulation in cardiomyocytes and consequently inhibits PKA phosphorylation of phospholamban and contractile responses in myocytes in vitro and in Langendorff perfused hearts. These data indicate that increased IR signaling, as occurs in hyperinsulinemic states, may directly impair βAR-regulated cardiac contractility. This β2AR-dependent IR and βAR signaling cross-talk offers a molecular basis for the broad interaction between these signaling cascades in the heart and other tissues or organs that may contribute to the pathophysiology of metabolic and cardiovascular dysfunction in insulin-resistant states.

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks.

Invasive fungal infections (IFI) cause considerable morbidity and mortality in pediatric patients. Serum biomarkers such as 1,3-beta-D glucan (BDG) and galactomannan (GM) have been evaluated for the IFI diagnosis. However, most evidence regarding their utility is derived from studies in adult oncology patients. This systematic review aimed to compare the diagnostic accuracy of BDG and GM individually or in combination for diagnosing IFI in pediatric patients. PubMed, CINAHL, Embase, and Cochrane Library were searched until March 2019 for diagnostic studies evaluating both serum GM and BDG for diagnosing pediatric IFI. The pooled diagnostic odds ratio (DOR), specificity and sensitivity were computed. Receiver operating characteristics (ROC) curve and area under the curve (AUC) were used for summarizing overall assay performance. Six studies were included in the meta-analysis. The summary estimates of sensitivity, specificity, pooled DOR, AUC of the GM assay for proven or probable IFI were 0.74, 0.76, 13.25, and 0.845. The summary estimates of sensitivity, specificity, pooled DOR, AUC of the BDG assay were 0.70, 0.69, 4.3, and 0.722. The combined predictive ability of both tests was reported in two studies (sensitivity: 0.67, specificity: 0.877). Four studies were performed in hematology-oncology patients, while two were retrospective studies from pediatric intensive care units (ICUs). In the subgroup of hematology-oncology patients, DOR of BDG remained similar at 4.25 but increased to 40.28 for GM. We conclude that GM and BDG have a modest performance for identifying IFI in pediatric patients. GM has a better accuracy over BDG. Combining both improves the specificity at the cost of sensitivity. [ABSTRACT FROM AUTHOR]

Prior studies have demonstrated associations between beta-adrenergic receptor (βAR) polymorphisms and left ventricular dysfunction-an important cause of allograft nonutilization for transplantation. We hypothesized that βAR polymorphisms predispose donor hearts to LV dysfunction after brain death. A total of 1043 organ donors managed from 2001-2006 were initially studied. The following βAR single nucleotide polymorphisms were genotyped: β1AR 1165C/G (Arg389Gly), β1AR 145A/G (Ser49Gly), β2AR 46G/A (Gly16Arg) and β2AR 79C/G (Gln27Glu). In multivariable regression analyses, the β2AR46 SNP was significantly associated with LV systolic dysfunction, with each minor allele additively decreasing the odds for LV ejection fraction 10 μg/kg/min of dopamine compared to those with the CC and GG genotypes. However, no significant associations were found between βAR SNPs and cardiac dysfunction in 364 donors managed from 2007-2008, perhaps due to changes in donor management, lack of power in this validation cohort, or the absence of a true association. βAR polymorphisms may be associated with cardiac dysfunction after brain death, but these relationships require further study in independent donor cohorts.

β-mannanase is one of the key enzymes to hydrolyze hemicellulose. At present, most β-mannanases are not widely applied because of their low enzyme activity and unsuitable enzymatic properties. In this work, a new β-mannanase from Enterobacter aerogenes was studied, which laid the foundation for its further application. Additionally, we will further perform directed evolution of the enzyme to increase its activity, improve its temperature and pH properties to allow it more applications in industry. A new β-mannanase (Man26E) from Enterobacter aerogenes was successfully expressed in Escherichia coli. Man26E showed about 40 kDa on SDS-PAGE gel. The SWISS-MODEL program was used to model the tertiary structure of Man26E, which presented a core (α/β)8-barrel catalytic module. Based on the binding pattern of CjMan26 C, Man26E docking Gal1Man4 was investigated. The catalytic region consisted of a surface containing four solvent-exposed aromatic rings, many hydrophilic and charged residues. Man26E displayed the highest activity at pH 6.0 and 55 °C, and high acid and alkali stability in a wide pH range (pH 4–10) and thermostability from 40 to 50 °C. The enzyme showed the highest activity on locust bean gum, and the Km and Vmax were 7.16 mg mL−1 and 508 U mg−1, respectively. This is the second β-mannanase reported from Enterobacter aerogenes B19. The β-mannanase displayed high enzyme activity, a relatively high catalytic temperature and a broad range of catalytic pH values. The enzyme catalyzed both polysaccharides and manno-oligosaccharides. [ABSTRACT FROM AUTHOR]

Background: Numerous studies have reported significantly elevated titers of serum autoantibody against the second extracellular loop of β1-adrenoceptor (β1-AA), a catecholamine-like substance with β1-adrenergic activity, in patients with heart failure. Although evidence demonstrates that this autoantibody may alter T cell proliferation and secretion, the role of T lymphocytes in heart failure induced by β1-AA remains unclear. The current study was designed to determine whether T cell disorder contributes to heart failure induced by β1-AA. Methods and Results: β1-AA monoclonal antibodies (β1-AAmAb) produced using the hybridoma technique were administered in wild-type mice or T lymphocyte deficiency nudes for 12 weeks. T lymphocytes from heart failure patients and neonatal cardiomyocytes were utilized in vitro. Mouse protein antibody array analysis was employed to detect the cytokines responsible for β1-AAmAb-induced heart failure. Compared to wild-type mice, T lymphocyte deficiency mice prevented cardiac function from getting worse, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. As shown by protein array, the serum level of interleukin (IL)-6 was significantly lower in the nude group as compared to wild-type after β1-AAmAb treatment. Mechanistic studies in vitro demonstrated that T lymphocyte culture supernatants stimulated by β1-AAmAb caused direct damage in the cardiomyocytes, and β1-AAmAb promoted proliferation of T lymphocytes isolated from patients with heart failure and increased IL-6 release. IL-6-specific siRNA virtually abolished cardiomyocyte apoptosis, suggesting that IL-6 may be a key cytokine released by T lymphocytes and responsible for β1-AAmAb-induced cardiac remodeling. Conclusions: Collectively, we demonstrate that β1-AAmAb-induced cardiac remodeling via mediating T lymphocyte disorder and releasing a variety of IL-6. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of nociceptin/orphanin FQ (N/OFQ) on ventricular arrhythmias induced by acute myocardial ischemia, and the role of β1-adrenergic receptor (β1-AR) thereof. Methods The rat model of acute myocardial ischemia was prepared by ligating the left anterior descending branch of the coronary artery. A total of 60 rats were randomly divided into 3 groups, namely sham-operation group (Sham group), coronary artery occlusion group (CAO group) and the pretreatment of N/OFQ receptor antagonist (UFP-101) group (U+CAO group). Ten rats were sacrificed at 15 minutes, and another ten rats were sacrificed at 1 hour after CAO. Electrocardiographic data were recorded. The expressions of β1-AR from myocardial cell membrane and whole cell were detected by Western blot assay. The expression of β1-AR mRNA was detected by RT-qPCR. Results Compared with Sham group, ischemic arrhythmias occurred in CAO group, and mainly occurred during 15 minutes after CAO. The pretreatment with UFP-101 can significantly decrease ischemic arrhythmias. At 15 min, compared with Sham group, the expressions of β1-AR and β1-AR mRNA in whole cell were downregulated, and the membrane β1-AR was up-regulated (all P＜0.05) in CAO group. Meanwhile, no significant differences were found in the expression of β1-AR and its mRNA in whole cell (all P＞0.05). The expression of membrane β1-AR was down-regulated (P＜0.05) in U+CAO group. Compared with the CAO group, the expressions of β1-AR and β1-AR mRNA in whole cell were up-regulated while the β1-AR in membrane was down-regulated (all P＜0.05) in U+CAO group. At 1 h, compared with Sham group, the expressions of β1-AR and β1-AR mRNA in the whole cell were up-regulated, and the membrane β1-AR was down-regulated (all P＜0.05) in CAO and U+CAO group. Conclusion Endogenous N/OFQ can regulate the expression of β1-AR in myocardial cell membrane and participate in the process of ischemic arrhythmia in rats. [ABSTRACT FROM AUTHOR]

Background: Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG). Methods: A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Results: Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. Conclusion: The mutations c.1456C < T (p.L486F) in MYOC and c.322G < A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.

β-mannanase is one of the key enzymes to hydrolyze hemicellulose. At present, most β-mannanases are not widely applied because of their low enzyme activity and unsuitable enzymatic properties. In this work, a new β-mannanase from Enterobacter aerogenes was studied, which laid the foundation for its further application. Additionally, we will further perform directed evolution of the enzyme to increase its activity, improve its temperature and pH properties to allow it more applications in industry. A new β-mannanase (Man26E) from Enterobacter aerogenes was successfully expressed in Escherichia coli. Man26E showed about 40 kDa on SDS-PAGE gel. The SWISS-MODEL program was used to model the tertiary structure of Man26E, which presented a core (α/β)8-barrel catalytic module. Based on the binding pattern of CjMan26 C, Man26E docking Gal1Man4 was investigated. The catalytic region consisted of a surface containing four solvent-exposed aromatic rings, many hydrophilic and charged residues. Man26E displayed the highest activity at pH 6.0 and 55 °C, and high acid and alkali stability in a wide pH range (pH 4–10) and thermostability from 40 to 50 °C. The enzyme showed the highest activity on locust bean gum, and the Km and Vmax were 7.16 mg mL−1 and 508 U mg−1, respectively. This is the second β-mannanase reported from Enterobacter aerogenes B19. The β-mannanase displayed high enzyme activity, a relatively high catalytic temperature and a broad range of catalytic pH values. The enzyme catalyzed both polysaccharides and manno-oligosaccharides.

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Beta-1,4-galactosyltransferase II is found to be associated with the alterations of tumor-related glycosylation. However, the clinical significance of beta-1,4-galactosyltransferase II in non-metastatic clear-cell renal cell carcinoma has not been reported up to now. Herein, our researches suggested that the expression level of beta-1,4-galactosyltransferase II was first found to be positively associated with tumor size, Fuhrman grade, lymphovascular invasion, rhabdoid differentiation, tumor necrosis and poor overall survival and recurrence-free survival of patients with non-metastatic clear-cell renal cell carcinoma, both in training set and validation set. Moreover, beta-1,4-galactosyltransferase II expression was identified as an independent adverse prognosticator for overall survival and recurrence-free survival of patients with non-metastatic clear-cell renal cell carcinoma. Ultimately, prognostic accuracy of the nomogram integrating beta-1,4-galactosyltransferase II with other independent prognostic parameters was dramatically improved for overall survival and recurrence-free survival of patients with non-metastatic clear-cell renal cell carcinoma. Taken together, beta-1,4-galactosyltransferase II is a potential independent adverse prognostic factor for postoperative recurrence and survival, which could be developed as a useful biomarker for non-metastatic clear-cell renal cell carcinoma by a series of further independent and retrospective studies, so as to help the postsurgical management of clear-cell renal cell carcinoma patients. [ABSTRACT FROM AUTHOR]

Beta-1,3-glucanases (EC 3.2.1.39), commonly known as pathogenesis-related (PR) proteins, play an important role not only in plant defense against fungal pathogens but also in plant physiological and developmental processes. However, only a limited number of sugarcane beta-1,3-glucanase genes have been isolated. In the present study, we identified and characterized a new beta-1,3-glucanase gene ScGluD2 (GenBank Acc No. KF664181) from sugarcane. An X8 domain was present at the C terminal region of ScGluD2, suggesting beta-1,3-glucan-binding function. Phylogenetic analysis showed that the predicted ScGluD2 protein was classified into subfamily D beta-1,3-glucanase. Localization of the ScGluD2 protein in the plasma membrane was determined by tagging it with green fluorescent protein. The expression of ScGluD2 was more up-regulated in sugarcane smut-resistant cultivars in the early stage (1 d or 3 d) than in the susceptible ones after being challenged by the smut pathogen, revealing that ScGluD2 may be involved in defense against the invasion of Sporisorium scitamineum. Transient overexpression of ScGluD2 in Nicotiana benthamiana leaves induced a defense response and exhibited antimicrobial action on the tobacco pathogens Pseudomonas solanacearum and Botrytis cinerea, further demonstrating that ScGluD2 was related to the resistance to plant pathogens. However, the transcripts of ScGluD2 partially increased (12 h) under NaCl stress, and were steadily up-regulated from 6 h to 24 h upon ABA, H2O2, and CdCl2 treatments, suggesting that ABA may be a signal molecule regulating oxidative stress and play a role in the salt and heavy metal stress-induced stimulation of ScGluD2 transcripts. Taken together, ScGluD2, a novel member of subfamily D beta-1,3-glucanase, was a stress-related gene of sugarcane involved in plant defense against smut pathogen attack and salt and heavy metal stresses.

Beta-1,3-glucanases (EC 3.2.1.39), commonly known as pathogenesis-related (PR) proteins, play an important role not only in plant defense against fungal pathogens but also in plant physiological and developmental processes. However, only a limited number of sugarcane beta-1,3-glucanase genes have been isolated. In the present study, we identified and characterized a new beta-1,3-glucanase gene ScGluD2 (GenBank Acc No. KF664181) from sugarcane. An X8 domain was present at the C terminal region of ScGluD2, suggesting beta-1,3-glucan-binding function. Phylogenetic analysis showed that the predicted ScGluD2 protein was classified into subfamily D beta-1,3-glucanase. Localization of the ScGluD2 protein in the plasma membrane was determined by tagging it with green fluorescent protein. The expression of ScGluD2 was more up-regulated in sugarcane smut-resistant cultivars in the early stage (1 or 3 days) than in the susceptible ones after being challenged by the smut pathogen, revealing that ScGluD2 may be involved in defense against the invasion of Sporisorium scitamineum. Transient overexpression of ScGluD2 in Nicotiana benthamiana leaves induced a defense response and exhibited antimicrobial action on the tobacco pathogens Pseudomonas solanacearum and Botrytis cinerea, further demonstrating that ScGluD2 was related to the resistance to plant pathogens. However, the transcripts of ScGluD2 partially increased (12 h) under NaCl stress, and were steadily up-regulated from 6 to 24 h upon ABA, H2O2, and CdCl2 treatments, suggesting that ABA may be a signal molecule regulating oxidative stress and play a role in the salt and heavy metal stress-induced stimulation of ScGluD2 transcripts. Taken together, ScGluD2, a novel member of subfamily D beta-1,3-glucanase, was a stress-related gene of sugarcane involved in plant defense against smut pathogen attack and salt and heavy metal stresses. [ABSTRACT FROM AUTHOR]

Mast cells are primary mediators of allergic inflammation. Beta-1,3- glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 μg/ml BG, 100 mg/ml peptidoglycan (PGN), or 10 μM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. [ABSTRACT FROM AUTHOR]

Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548, was cloned into the Streptomyces/Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in S. lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 and 16.1 mg/mL, respectively. The Vmax values toward Avicel and filter paper were 0.432 and 0.084 μM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co2+, Cu2+, Ni2+ and Mn2+ (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-β-cellobiosidase that hydrolyzes cellulose into cellobiose. [ABSTRACT FROM AUTHOR]

1,3-ß-D-glucan ( BDG) is increasingly used to diagnose invasive fungal infections ( IFI), although false positive results are a concern. To evaluate the potential interaction of blood products with the BDG assay, human albumin ( HA), fresh frozen plasma ( FFP), undiluted platelet transfusion ( UPT) and packed red blood cells ( PRBC) were tested for their BDG content using two different b-D-glucan tests. UPTs tested negative, FFP, PBRC and HA tested positive for BDG. In serial dilution, BDG concentration correlated with blood product concentration. To investigate the clinical impact of blood product transfusions, we measured BDG levels before and after the transfusion in three patients (2 PRBC, 1 HA). In the patients receiving PRBC transfusions, BDG values increased from 13 and 17 pg ml−1 to 183 and 361 pg ml−1, the HA transfusion increased the serum level from 42 to 58 pg ml−1. BDG concentrations measured in blood products can be used to predict false positive BDG results. [ABSTRACT FROM AUTHOR]