PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

Background: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G(q)-and G(s)-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.Methods: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.Results: The beta(2)-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.Conclusion: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p

Functional Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia malayi Using RNA Interference

Filarial nematodes are an important group of human pathogens, causing lymphatic filariasis and onchocerciasis, and infecting around 150 million people throughout the tropics with more than 1.5 billion at risk of infection. Control of filariasis currently relies on mass drug administration (MDA) programs using drugs which principally target the microfilarial life-cycle stage. These control programs are facing major challenges, including the absence of a drug with macrofilaricidal or permanent sterilizing activity, and the possibility of the development of drug-resistance against the drugs available. Cysteine proteases are essential enzymes which play important roles in a wide range of cellular processes, and the cathepsin-like cysteine proteases have been identified as potential targets for drug or vaccine development in many parasites. Here we have studied the function of several of the cathepsin-like enzymes in the filarial nematode, B. malayi, and demonstrate that these cysteine proteases are involved in the development of embryos, show similar functions to their counterparts in C. elegans, and therefore, provide an important target for future drug development targeted to eliminate filariasis

Long-Term Persistance of the Pathophysiologic Response to Severe Burn Injury

Main contributors to adverse outcomes in severely burned pediatric patients are profound and complex metabolic changes in response to the initial injury. It is currently unknown how long these conditions persist beyond the acute phase post-injury. The aim of the present study was to examine the persistence of abnormalities of various clinical parameters commonly utilized to assess the degree hypermetabolic and inflammatory alterations in severely burned children for up to three years post-burn to identify patient specific therapeutic needs and interventions. Nine-hundred seventy-seven severely burned pediatric patients with burns over 30% of the total body surface admitted to our institution between 1998 and 2008 were enrolled in this study and compared to a cohort non-burned, non-injured children. Demographics and clinical outcomes, hypermetabolism, body composition, organ function, inflammatory and acute phase responses were determined at admission and subsequent regular intervals for up to 36 months post-burn. Statistical analysis was performed using One-way ANOVA, Student's t-test with Bonferroni correction where appropriate with significance accepted at p<0.05. Resting energy expenditure, body composition, metabolic markers, cardiac and organ function clearly demonstrated that burn caused profound alterations for up to three years post-burn demonstrating marked and prolonged hypermetabolism, p<0.05. Along with increased hypermetabolism, significant elevation of cortisol, catecholamines, cytokines, and acute phase proteins indicate that burn patients are in a hyperinflammatory state for up to three years post-burn p<0.05. Severe burn injury leads to a much more profound and prolonged hypermetabolic and hyperinflammatory response than previously shown. Given the tremendous adverse events associated with the hypermetabolic and hyperinflamamtory responses, we now identified treatment needs for severely burned patients for a much more prolonged time

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Quantifying Vegetation Biophysical Variables from Imaging Spectroscopy Data: A Review on Retrieval Methods

An unprecedented spectroscopic data stream will soon become available with forthcoming Earth-observing satellite missions equipped with imaging spectroradiometers. This data stream will open up a vast array of opportunities to quantify a diversity of biochemical and structural vegetation properties. The processing requirements for such large data streams require reliable retrieval techniques enabling the spatiotemporally explicit quantification of biophysical variables. With the aim of preparing for this new era of Earth observation, this review summarizes the state-of-the-art retrieval methods that have been applied in experimental imaging spectroscopy studies inferring all kinds of vegetation biophysical variables. Identified retrieval methods are categorized into: (1) parametric regression, including vegetation indices, shape indices and spectral transformations; (2) nonparametric regression, including linear and nonlinear machine learning regression algorithms; (3) physically based, including inversion of radiative transfer models (RTMs) using numerical optimization and look-up table approaches; and (4) hybrid regression methods, which combine RTM simulations with machine learning regression methods. For each of these categories, an overview of widely applied methods with application to mapping vegetation properties is given. In view of processing imaging spectroscopy data, a critical aspect involves the challenge of dealing with spectral multicollinearity. The ability to provide robust estimates, retrieval uncertainties and acceptable retrieval processing speed are other important aspects in view of operational processing. Recommendations towards new-generation spectroscopy-based processing chains for operational production of biophysical variables are given

Analysis are of the hidden properties of the macromolecular system as an example of the reaction centers of bacteria Rhodobacter sphaeroides

Relevant: Is the study of the response of biological macromolecules to external stimuli. Often the reaction of macromolecules has an effect of structural self-regulation. In this case, their reaction is not only external infl uence, but also the spatial-temporal motions of the macromolecule. In this situation deserves the attention of electronic-conformational interactions of macromolecules. As objects are isolated Reaction Centers (RC) of bacteria Rhodobacter sphaeroides, the structure of which is well studied. During prolonged illumination, the RC occurs intramolecular electron transfer of RC, the kinetics of which has expressed as a sum of three different exponential functions with negative values decrements. In this case, can considered two models of electron transfer with variable or constant with time of the kinetic parameters.</p