Academic literature on the topic 'Camp #21 (Cascade Locks, Or.)'

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as α subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Δbcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Δbac, lesion development of Δbcpka1 and ΔbcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Δbac, Δbcpka1 and ΔbcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Δbcpka1 and ΔbcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Gα subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.

Introduction. Converging evidence suggests that PDE-4 (phosphodiesterase subtype 4) plays a crucial role in regulating cognition via the PDE-4-cAMP cascade signaling involving phosphorylated cAMP response element binding protein (CREB).Objective. The primary endpoint was to examine the neurocognitive effects of extractSceletium tortuosum(Zembrin) and to assess the safety and tolerability of Zembrin in cognitively healthy control subjects.Method. We chose the randomized double-blind placebo-controlled cross-over design in our study. We randomized normal healthy subjects (totaln=21) to receive either 25 mg capsule Zembrin or placebo capsule once daily for 3 weeks, in a randomized placebo-controlled 3-week cross-over design. We administered battery of neuropsychological tests: CNS Vital Signs and Hamilton depression rating scale (HAM-D) at baseline and regular intervals and monitored side effects with treatment emergent adverse events scale.Results. 21 subjects (mean age: 54.6 years ± 6.0 yrs; male/female ratio: 9/12) entered the study. Zembrin at 25 mg daily dosage significantly improved cognitive set flexibility (P<0.032) and executive function (P<0.022), compared with the placebo group. Positive changes in mood and sleep were found. Zembrin was well tolerated.Conclusion. The promising cognitive enhancing effects of Zembrin likely implicate the PDE-4-cAMP-CREB cascade, a novel drug target in the potential treatment of early Alzheimer’s dementia. This trial is registered with ClinicalTrials.govNCT01805518.

To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed. Other modes of GPK activation were not apparent, for the enzyme in crude extracts was stimulated only by incubation with cAMP and did not respond to cGMP or Ca2+ + phorbol 12-myristate 13-acetate. The data indicate that the cAMP-activated, PKA- and GPK-mediated cascade is key to the activation of glycogenolysis in both the skeletal muscle and liver during burst swimming exercise by trout.Key words: exercise, glycogen phosphorylase kinase, protein kinase A, cAMP, Oncorhynchus mykiss, control of glycogenolysis.

Prenatal hypoxia leads to an increased risk of adult cardiovascular disease. We have previously demonstrated a programming effect of prenatal hypoxia on the cardiac β-adrenergic (βAR) response. The aim of this study was to determine 1) whether the decrease in βAR sensitivity in prenatally hypoxic 5-wk old chicken hearts is linked to changes in β1AR/β2ARs, Gαi expression and cAMP accumulation and 2) whether prenatal hypoxia has an effect on heart function in vivo. We incubated eggs in normoxia (N, 21% O2) or hypoxia from day 0 (H, 14% O2) and raised the posthatchlings to 5 wk of age. Cardiac β1AR/β2ARs were assessed through competitive binding of [3H]CGP-12177 with specific β1AR or β2AR blockers. Gαs and Gαi proteins were assessed by Western blot and cAMP accumulation by ELISA. Echocardiograms were recorded in anesthetized birds to evaluate diastolic/systolic diameter and heart rate and tissue sections were stained for collagen. We found an increase in relative heart mass, β1ARs, and Gαs in prenatally hypoxic hearts. cAMP levels after isoproterenol stimulation and collagen content was not changed in H compared with N, but in vivo echocardiograms showed systolic contractile dysfunction. The changes in βAR and G protein subtypes may be indicative of an early compensatory stage in the progression of cardiac dysfunction, further supported by the cardiac hypertrophy and systolic contractile dysfunction. We suggest that it is not the changes in the proximal part of the βAR system that causes the decreased cardiac contractility, but Ca2+ handling mechanisms further downstream in the βAR signaling cascade.

Primary cultures of progenitor and immature rat Leydig cells were established from the testes of 21- and 35-d-old rats, respectively. The cell population remained homogeneous after 4–6 d in culture as judged by staining for 3β-hydroxysteroid dehydrogenase, but the cells were unable to bind 125I-human chorionic gonadotropin (hCG) or to respond to hCG with classical LH receptor (LHR)-mediated responses, including cAMP and inositol phosphate accumulation, steroid biosynthesis, or the phosphorylation of ERK1/2. Infection of primary cultures with recombinant adenovirus coding for β-galactosidase showed that approximately 65% of the cells are infected. Infection with adenovirus coding for the human LHR (hLHR) allowed for expression of the hLHR at a density of approximately 25,000 receptors per cell and allowed the cells to respond to hCG with increases in cAMP and inositol phosphate accumulation, steroid biosynthesis, and the phosphorylation of ERK1/2. Although progenitor and immature cells were able to respond to hCG with an increase in progesterone, only the immature cells responded with an increase in testosterone. In addition to these classical LHR-mediated responses, the primary cultures of progenitor or immature rat Leydig cells expressing the recombinant hLHR proliferated robustly when incubated with hCG, and this proliferative response was sensitive to an inhibitor of ERK1/2 phosphorylation. These studies establish a novel experimental paradigm that can be used to study the proliferative response of Leydig cells to LH/CG. We conclude that activation of the LHR-provoked Leydig cell proliferation requires activation of the ERK1/2 cascade.

The effects of cold exposure (7 days, 5 degrees C) and cold acclimation (21 days, 5 degrees C) on the regulation of lipolysis were investigated in adipocytes isolated from epididymal fat pads of rats. Catecholamines stimulated lipolysis in an affinity sequence typical of the beta 1-adrenoceptor subtype: one-half maximum velocity (1/2 Vmax) isoproterenol (35 nM) much greater than 1/2 Vmax norepinephrine (150 nM) approximately 1/2 Vmax epinephrine (200 nM). Cold exposure markedly decreased the sensitivity (1/2 Vmax) and the responsiveness (Vmax) of the adipocytes to the lipolytic action of catecholamines. Addition of adenosine deaminase to fat cells isolated from cold-exposed rats did not normalize the lipolytic activity, suggesting that extracellular adenosine was not responsible for the obtunded lipolysis. This effect of cold exposure was transient as the lipolytic response to catecholamines was normal in fully cold-acclimated animals. Remarkably, the responsiveness of adipocytes to the lipolytic action of glucagon (200 nM) and adrenocorticotropic hormone (ACTH, 1 microM) progressively increased during cold acclimation. Adipocyte lipolytic response to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline was normal in cold-exposed rats, indicating that the lipolytic defect resides at an early step in the lipolytic cascade (pre-cAMP). On the other hand, the antilipolytic effect of insulin on norepinephrine-induced lipolysis significantly decreased during cold acclimation, particularly at physiological levels of insulin (nanomolar level). These results demonstrate that the transient decrease in the lipolytic action of catecholamines observed during cold acclimation is compensated by 1) an increased responsiveness of adipocytes to glucagon and ACTH and 2) by a decreased effectiveness of insulin to induce antilipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract Abstract 226 Umbilical cord blood transplantation (UCBT) has extended the availability of hematopoietic stem cell transplantation (HSCT) to patients without compatible adult stem cell donors. However, prolonged time to engraftment, delayed immunologic reconstitution and late memory T cell skewing compromise the favorable outcome of UCBT. Studies in zebrafish and mouse models have shown that the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (PGE2) increases HSC number, homing and engraftment by modifying the Wnt signaling cascade through cAMP/PKA-mediated stabilization of β-catenin and expression of Wnt target genes. We performed a Phase Ib clinical trial of double UCBT (dUCBT), using one untreated and one ex vivo PGE2-treated UCB unit, to determine safety of this uproach. 12 subjects with hematologic malignancies were enrolled (median age 58.1 years (19.8–66.5)). The median time to engraftment was 17.5 days, superior to a 21 day median for a historic control group (n=53; p=0.045). The PGE2-UCB was the dominant source of hematopoiesis in 10 of 12 subjects (p=0.039) with full T cell chimerism as early as 13 days after dUCBT, suggesting that PGE2 regulated the outcome of UCBT by T cell-dependent mechanisms. Here we examined whether PGE2 might alter the properties of UCB T cells thereby affecting T cell reconstitution after UCBT. We determined that EP2 and EP4 receptors were constitutively expressed in UCB T cells and mediated increase of intracellular cAMP in response to PGE2. PGE2 modulated the Wnt/β-catenin pathway in UCB T cells as determined by upregulation of β-catenin and expression of Wnt target genes including Lef1, Tcf7 and Runx1. Similarly to pharmacologic activation of Wnt/β-catenin signaling by the Gsk3β inhibitor TWS119, PGE2 inhibited proliferation of UCB T cells in response to stimulation via TCR/CD3 and CD28. Under these conditions, a significant proportion of T cells expressed naïve immunophenotypic features with high levels of CD45RA, CD62L and CD127. To examine whether these PGE2-mediated effects on UCB T cells might have in vivo implications we assessed reconstitution of T lymphocytes in PGE2-UCBT recipients and compared the pattern of recovery with that of dUCBT recipients without PGE2 treatment. The numbers of total CD3+ T lymphocytes, CD4+ and CD8+ T subsets remained significantly lower (p=0.036) in PGE2-UCBT recipients during the first year after UCBT. In contrast, T cell receptor excisions circles (TRECs), a marker of naïve post-thymic T cells and indicator of thymic function, were substantially higher in PGE2-UCBT recipients and never reached the nadir observed in dUCBT recipients without PGE2, who had values below the limit of detection through 100 days. Importantly, PGE2-UCBT recipients displayed potent antiviral immunity as determined by the reduced incidence of CMV viremia and no cases of EBV-mediated posttransplant lymphoproliferative disorder (PTLD), which caused significant morbidity and mortality in dUCBT recipients without PGE2. In mouse models Wnt/β-catenin signaling promotes the generation of a central memory/stem cell memory T cell population with naïve immunophenotypic features but potent immune function. To examine whether such PGE2-mediated Wnt/β-catenin imprinting might be evident in PGE2-UCBT recipients, we examined expression of the Wnt/β-catenin target Eomes, a transcription factor that links the long-term memory CD8+ T cells to effector potency and protective immunity. Real time qPCR revealed that expression of Eomes was significantly elevated in the PBMCs of PGE2-UCBT recipients compared to control UCBT recipients. Consistent with the role of Wnt/β-catenin to maintain a central memory/stem cell memory phenotype in CD8+ cells, there was an increased fraction of CD62L+ cells within the CD8+ populations in recipients of PGE2-UCBT. Our in vitro and in vivo findings indicate that PGE2-UCB treatment improves HSC engraftment while preserving a naïve T cell compartment and favoring the generation of long-lived memory CD8+ cells via Wnt-mediated gene programming. This novel treatment might significantly improve the outcome of UCBT where delayed engraftment and impaired immunity are serious causes of morbidity and mortality. Disclosures: No relevant conflicts of interest to declare.

Objective: Recent evidence suggests an important role for cAMP-dependent pathways in modulation of innate immune function. Phosphodiesterase 4 (PDE4) is widely expressed in innate immune cells such as macrophages/dendritic cells with potent anti-inflammatory effects on pharmacologic inhibition of the enzyme. We investigated the importance of PDE4 in diet-induced obesity (DIO) and hypothesized that PDE4 inhibition will improve insulin sensitivity and reduce inflammation. Methods and Results: PDE4 was upregulated in both visceral and subcutaneous (SubQ) white adipose tissue (WAT) in DIO mice (12 weeks of high-fat diet, HFD, 60% fat) compared to normal-chow diet (NCD) mice (↑4∼10-folds, p<0.01). The degree of expression was correlated with macrophage infiltration in stromal vascular fraction from WAT (CD11b + F4/80 + cells, r=0.56, p<0.05). Treatment with Roflumilast (3mg/kg/day), a high affinity inhibitor of PDE4 (IC 50 0.39 nM) versus vehicle control (n=6∼10 in each group) for 21 days concomitant with HFD, resulted in rapid and substantial weight loss (↓45.8% fat content), enhanced thermogenesis [(∼20% higher oxygen consumption and heat production, 0.7∼1.1°C higher core body temperature in a cold environment (4°C)], brown adipose reprogramming, improvement in insulin resistance (HOMA-IR ↓ from 0.69±0.04 to 0.44±0.01, p<0.01) and hepatic steatosis. These changes were paralleled by increased alternative macrophage activation (Altf), reduced inflammation in WAT [↑CD206 and CD301 by flow cytometry with ↓ TNF/IL-6 gene expression] and activation of thermogenic genes in brown adipose tissue. In-vitro treatment of mouse bone marrow-derived macrophages (BMDM) promoted Altf and increased expression of tyrosine hydroxylase (↑2.5 folds) and catecholamines secretion. Additional experiments with agents that augment/reduce intracellular cAMP/EPAC/AMPK revealed an essential role for this cascade in Altf activation and catecholamine release. Conclusions: PDE4 antagonism improves obese diabetic symptoms through convergent pathways involving Altf activation and enhancing thermogenesis via cAMP dependent modulation of macrophage catecholamine release.