Search

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association. We are grateful to Xuexun Fang (Laboratory of Molecular Enzymology and Enzyme Engineering of the Ministry of Education, Jilin University, China) for providing the pHSIE vector.

The term "IgY technology" was introduced in the literature in the mid 1990s to describe a procedure involving immunization of avian species, mainly laying hens and consequent isolation of the polyclonal IgYs from the "immune" egg yolk (thus avoiding bleeding and animal stress). IgYs have been applied to various fields of medicine and biotechnology. The present article will deal with specific aspects of IgY technology, focusing on the currently reported methods for developing, isolating, evaluating and storing polyclonal IgYs. Other topics such as current information on isolation protocols or evaluation of IgYs from different avian species are also discussed. Specific advantages of IgY technology (e.g., novel antibody specificities that may emerge via the avian immune system) will also be discussed. Recent in vitro applications of polyclonal egg yolk-derived IgYs to the field of disease diagnosis in human and veterinary medicine through in vitro immunodetection of target biomolecules will be presented. Moreover, ethical aspects associated with animal well-being as well as new promising approaches that are relevant to the original IgY technology (e.g., development of monoclonal IgYs and IgY-like antibodies through the phage display technique or in transgenic chickens) and future prospects in the area will also be mentioned.

Introduction Resistive breathing (RB), the pathophysiologic hallmark of chronic obstructive pulmonary disease (COPD), especially during exacerbations, is associated with significant inflammation and mechanical stress on the lung. Mechanical forces are implicated in the progression of emphysema that is a major pathologic feature of COPD. We hypothesized that resistive breathing exacerbates emphysema. Methods C57BL/6 mice were exposed to 0.75 units of pancreatic porcine elastase intratracheally to develop emphysema. Resistive breathing was applied by suturing a nylon band around the trachea to reduce surface area to half for the last 24 or 72 hours of a 21-day time period after elastase treatment in total. Following RB (24 or 72 hours), lung mechanics were measured and bronchoalveolar lavage (BAL) was performed. Emphysema was quantified by the mean linear intercept (Lm) and the destructive index (DI) in lung tissue sections. Results Following 21 days of intratracheal elastase exposure, Lm and DI increased in lung tissue sections [Lm (μm), control 39.09±0.76, elastase 62.05±2.19, p=0.003 and DI, ctr 30.95±2.75, elastase 73.12±1.75, p

Dimitrios Toumpanakis,1 Athanasia Chatzianastasiou,1 Vyronia Vassilakopoulou,1 Eleftheria Mizi,1 Maria Dettoraki,1 Fotis Perlikos,1 Georgia Giatra,1,2 Nikolaos Mikos,3 Stamatios Theocharis,4 Theodoros Vassilakopoulos1,2 1“Marianthi Simou” Applied Biomedical Research and Training Center, Evangelismos Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 2 3rd Department of Critical Care Medicine, Evgenideio Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; 3Allergology Department, Laiko General Hospital, Athens, Greece; 4First Department of Pathology, Medical School, National and Kapodistrian University of Athens, Athens, GreeceCorrespondence: Dimitrios Toumpanakis, Email dtoumpanakis@yahoo.grIntroduction: TRPV4 channels are calcium channels, activated by mechanical stress, that have been implicated in the pathogenesis of pulmonary inflammation. During resistive breathing (RB), increased mechanical stress is imposed on the lung, inducing lung injury. The role of TRPV4 channels in RB-induced lung injury is unknown.Materials and Methods: Spontaneously breathing adult male C57BL/6 mice were subjected to RB by tracheal banding. Following anaesthesia, mice were placed under a surgical microscope, the surface area of the trachea was measured and a nylon band was sutured around the trachea to reduce area to half. The specific TRPV4 inhibitor, HC-067047 (10 mg/kg ip), was administered either prior to RB and at 12 hrs following initiation of RB (preventive) or only at 12 hrs after the initiation of RB (therapeutic protocol). Lung injury was assessed at 24 hrs of RB, by measuring lung mechanics, total protein, BAL total and differential cell count, KC and IL-6 levels in BAL fluid, surfactant Protein (Sp)D in plasma and a lung injury score by histology.Results: RB decreased static compliance (Cst), increased total protein in BAL (p < 0.001), total cell count due to increased number of both macrophages and neutrophils, increased KC and IL-6 in BAL (p < 0.001 and p = 0.01, respectively) and plasma SpD (p < 0.0001). Increased lung injury score was detected. Both preventive and therapeutic HC-067047 administration restored Cst and inhibited the increase in total protein, KC and IL-6 levels in BAL fluid, compared to RB. Preventive TRPV4 inhibition ameliorated the increase in BAL cellularity, while therapeutic TRPV4 inhibition exerted a partial effect. TRPV4 inhibition blunted the increase in plasma SpD (p < 0.001) after RB and the increase in lung injury score was also inhibited.Conclusion: TRPV4 inhibition exerts protective effects against RB-induced lung injury.Keywords: resistive breathing, TRPV4, lung permeability, pulmonary inflammation

Proinflammatory cytokines like interleukin-1β (IL-1β) affect the control of breathing. Our aim is to determine the effect of the anti-inflammatory cytokine IL-10 οn the control of breathing. IL-10 knockout mice (IL-10−/−, n = 10) and wild-type mice (IL-10+/+, n = 10) were exposed to the following test gases: hyperoxic hypercapnia 7% CO2-93% O2, normoxic hypercapnia 7% CO2-21% O2, hypoxic hypercapnia 7% CO2-10% O2, and hypoxic normocapnia 3% CO2-10% O2. The ventilatory function was assessed using whole body plethysmography. Recombinant mouse IL-10 (rIL-10; 10 μg/kg) was administered intraperitoneally to wild-type mice ( n = 10) 30 min before the onset of gas challenge. IL-10 was administered in neonatal medullary slices (10–30 ng/ml, n = 8). We found that IL-10−/−mice exhibited consistently increased frequency and reduced tidal volume compared with IL-10+/+mice during room air breathing and in all test gases (by 23.62 to 33.2%, P < 0.05 and −36.23 to −41.69%, P < 0.05, respectively). In all inspired gases, the minute ventilation of IL-10−/−mice was lower than IL-10+/+(by −15.67 to −22.74%, P < 0.05). The rapid shallow breathing index was higher in IL-10−/−mice compared with IL-10+/+mice in all inspired gases (by 50.25 to 57.5%, P < 0.05). The intraperitoneal injection of rIL-10 caused reduction of the respiratory rate and augmentation of the tidal volume in room air and also in all inspired gases (by −12.22 to −29.53 and 32.18 to 45.11%, P < 0.05, respectively). IL-10 administration in neonatal rat ( n = 8) in vitro rhythmically active medullary slice preparations did not affect either rhythmicity or peak amplitude of hypoglossal nerve discharge. In conclusion, IL-10 may induce a slower and deeper pattern of breathing.

Introduction: Resistive breathing (RB) is the hallmark of the pathophysiology of obstructive airway diseases. RB is associated with increased mechanical stress on the lung and induces lung injury in previously healthy animals.TRPV4 are mechanosensitive channels, implicated recently in the pathogenesis of lung injury. Aim: To investigate the role of TRPV4 channels in RB-induced lung injury. Methods: Spontaneously breathing C57BL/6 mice were subjected to RB by tracheal banding. Anaesthetized mice were placed under a surgical microscope and the surface area of the trachea was reduced to half by placement of a nylon band around the trachea. TRPV4 antagonist, HC-067047 (10 mg/kg ip), was administered either prior to RB and at 12 hrs following initiation of RB (preventive) or only at 12 hrs after the initiation of RB (therapeutic strategy). Following 24 hrs of RB in total, a static pressure-volume curve was obtained. Total protein, KC and IL-6 levels were measured in BAL fluid. Surfactant Protein (Sp)D in plasma was measured and a lung injury score was estimated by histology. Results: RB decreased static compliance (Cst) (p=0.016), increased total protein in BAL (p Conclusion: TRPV4 inhibition protects against RB-induced lung injury.

The development of peptide-based vaccines for treating human neurodegenerative diseases has been the eventual aim of many research endeavors, although no active immunotherapies have been approved for clinical use till now. A typical example of such endeavors is the effort to develop vaccines for Alzheimer’s disease based on the beta-amyloid peptide, which continues to be intensively investigated despite previous setbacks. In this paper, recent developments in peptide-based vaccines which target beta-amyloid as well as tau protein and α-synuclein are presented. Particular focus has been directed toward peptide epitopes and formulation systems selected/developed and employed to enhance vaccine efficacy and safety. Results from both, human clinical trials and animal preclinical studies conducted mainly in transgenic mice have been included. Future perspectives on the topic are also briefly discussed.

Dimitrios Toumpanakis,1,2 Konstantinos Loverdos,1,2 Vassiliki Tzouda,1,2 Vyronia Vassilakopoulou,1,2 Eleni Litsiou,1,2 Christina Magkou,3 Vassiliki Karavana,1,2 Michael Pieper,4 Theodoros Vassilakopoulos1,2 1First Critical Care Department, Pulmonary Unit, National and Kapodistrian University of Athens Medical School, Evangelismos General Hospital, 2George P. Livanosand Marianthi Simou Laboratories, Thorax Foundation, 3Department of Pathology, Evangelismos General Hospital, Athens, Greece; 4Boehringer Ingelheim Pharma GmbH & Co. KG Div. Research Germany, Biberach, Germany Introduction: Resistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown.Methods: Adult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1β and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically.Results: In all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1β and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1β, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1β and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1β and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations.Conclusion: Tiotropium inhalation attenuates RB-induced pulmonary inflammation. Keywords: resistive breathing, inflammation, tiotropium bromide

Study Question\ud Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility?\ud Summary Answer\ud Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols.\ud What is Known Already\ud Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCζ antibody specificity and detection protocols.\ud Study Design, Size Duration\ud Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls.\ud Participants/Materials, Setting, Methods\ud Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1–0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats.\ud Main Results and the Role of Chance\ud Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCζ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCζ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males.\ud Limitations, Reasons for Caution\ud Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status.\ud Wider Implications of the Findings\ud We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCζ appears to be kept in an inactive form in the sperm.

Prothymosin alpha (ProTα) is a highly conserved polypeptide (109 amino acids in humans) with diagnostic and therapeutic potential; ProTα exerts intra- and extra-cellular biological functions associated with cell proliferation, apoptosis and immune regulation, while it has been suggested to act as a damage-associated molecular pattern (DAMP) or alarmin. In this work, chicken polyclonal anti-ProTα antibodies that had been developed several years ago were immunochemically evaluated and proven to retain immunoreactivity for ProTα, with remarkable thermal and pH stability. Moreover, the antibodies showed practically no cross-reactivity with a series of ProTα-fragments, eventually intracellularly produced -such as ProTα[1-28] (also known as Tα1) and ProTα[100-109], which exert per se biological activity and might be present in biological samples along with the intact molecule, being therefore highly specific for whole-length ProTα. Based on the above antibodies (IgYs-3e), a highly specific competitive ProTα-ELISA with well-studied analytical characteristics (intra- and inter-assay CVs: ≤5% and ≤12%, respectively, limit of detection: 2.1 ng/mL, recovery: 88–104%) was developed. The new ProTα-ELISA was applied to the analysis of supernatants of HeLa cells driven to necrosis; intact ProTα was measured in cell culture supernatants, at levels that seemed to depend on % cell necrosis., Cell biology; Immunology; Proteins; Biochemistry; Cell necrosis; ELISA; HeLa cell culture supernatants; Immunoglobulins Y (IgY); Prothymosin alpha (ProTα)

Background: Our research group has shown that resistive breathing (RB) induces pulmonary inflammation in previously healthy animals. We hypothesized that RB would exacerbate pulmonary inflammation and emphysema, when superimposed on COPD animal models. Aim and Objective: To investigate the synergistic effect of RB on pulmonary inflammation and emphysema, when combined with experimental models of COPD. Methods: C57BL/6 mice were exposed to cigarette smoke (CS) for 1 or 6 months. In another group, mice were exposed to 0.75 units of elastase (PPE) intratracheally. Resistive breathing was applied by suturing a nylon band around the trachea to reduce surface area to half, for 24 hours following CS exposure or for the last 24 or 72 hours of 21 days after elastase treatment in total. Following RB, lung mechanics were measured and bronchoalveolar lavage (BAL) was performed. Emphysema was quantified by the Mean Linear (Lm) and the Destructive index (DI) in lung tissue sections. Surfactant Protein (SP)-D plasma levels were measured. Results: CS exposure for 1 and 6 months increased BAL cell number (p Conclusion: RB exacerbates pulmonary inflammation and emphysema in COPD animal models.

Ryanodine receptor 2 (RyR2) is a large transmembrane calcium (Ca2+) release channel that mediates Ca2 release from the sarcoplasmic reticulum to activate cardiac muscle contraction. Calmodulin (CaM) regulation of RyR2 is essential for normal cardiac function. A number of linear fragments of RyR2 have been reported as potential CaM-binding sequences. The sequence 3583-3603aa of human RyR2, which is highly conserved among mammalian isoforms, has been identified as a CaM-binding site in almost all relevant studies and therefore this region is considered as a well-established CaM-binding domain of RyRs. Besides 3583-3603aa region, other RyR2 regions have been also reported as potential CaM-binding sequences. Herein, we used recombinant wild-type CaMprotein and isothermal titration calorimetry (ITC) experiments to screen a number of RyR2-specific synthetic peptides corresponding to the region 4240-4277aa of RyR2, which has been previously proposed as a putative CaM-binding RyR2 region. From all the synthetic peptides screened, a peptide corresponding to 4255-4271aa region of human RyR2 was found to interact with significant affinity with RyR2, in the presence and absence of Ca2+ (Kd values 0.60 and 16.58 μM, respectively). Moreover, investigation of the interaction of four arrhythmogenic CaM mutants (N98I, D132E, D134H and Q136P) with this synthetic peptide, as well as the peptide corresponding to the well-established CaM-binding domain of RyR2 (3583-3603aa), revealed that all mutants show disparate binding properties to these two RyR2 peptides, which have been previously proposed to contribute to a putative intra-subunit CaM-binding pocket. Our findings extend our previous observations suggesting that CaM mutations may trigger arrhythmogenic cardiac disease by altering both intrinsic Ca2+-binding, as well as by dysregulating RyR2-mediated Ca2+ release via defective interaction of CaM with a distinct CaM-binding pocket that multiple RyR2 regions might contribute.

Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with strenuous contractions of the inspiratory muscles and increased negative intrathoracic pressures that act as an injurious stimulus to the lung. We have shown that IRB induces pulmonary inflammation in healthy animals. p38 kinase is activated in the lung under stress. We hypothesized that p38 is activated during IRB and contributes to IRB-induced pulmonary inflammation. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve. Resistance was connected to the inspiratory port to provoke a peak tidal inspiratory pressure 50% of maximum. Following 3 and 6 h of IRB, respiratory system mechanics were measured and bronchoalveolar lavage (BAL) was performed. Phosphorylated p38, TNF-α, and MIP-2α were detected in lung tissue. Lung injury was estimated histologically. SB203580 (p38 inhibitor) was administered prior to IRB (1 mg kg−1). Six hours of IRB increased phosphorylated p38 in the lung, compared with quietly breathing controls (p = 0.001). Six hours of IRB increased the numbers of macrophages and neutrophils (p = 0.01 and p = 0.005) in BAL fluid. BAL protein levels and lung elasticity increased after both 3 and 6 h IRB. TNF-α and MIP-2α increased after 6 h of IRB (p = 0.01 and p

Background Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury. Methods Anaesthetized, tracheostomized adult rats breathed spontaneously through a 2-way non-rebreathing valve. Resistance was added to the inspiratory port to provide a peak tidal inspiratory pressure of 50% of maximum (inspiratory resistive breathing). Activation of Src and ERK1/2 in the lung was estimated during IRB. Following 6 h of IRB, respiratory system mechanics were measured by the forced oscillation technique and bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. IL-1b and MIP-2a protein levels were measured in lung tissue samples. Wet lung weight to total body weight was measured and Evans blue dye extravasation was estimated to measure lung permeability. Lung injury was evaluated by histology. The Src inhibitor, PP-2 or the inhibitor of ERK1/2 activation, PD98059 was administrated 30 min prior to IRB. Results Src kinase was activated 30 min after the initiation of IRB. Src inhibition ameliorated the increase in BAL cellularity after 6 h IRB, but not the increase of IL-1β and MIP-2a in the lung. The increase in BAL total protein and lung injury score were not affected. The increase in tissue elasticity was partly inhibited. Src inhibition blocked ERK1/2 activation at 3 but not at 6 h of IRB. ERK1/2 inhibition ameliorated the increase in BAL cellularity after 6 h of IRB, blocked the increase of IL-1β and returned Evans blue extravasation and wet lung weight to control values. BAL total protein and the increase in elasticity were partially affected. ERK1/2 inhibition did not significantly change total lung injury score compared to 6 h IRB. Conclusions Src and ERK1/2 are activated in the lung following IRB and participate in IRB-induced lung injury.

Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca2 + channel complex that mediates Ca2 + efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation–contraction coupling and defective CaM–RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca2 +-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca2 +-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca2 +-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [3H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca2 +-binding as well as defective interaction with RyR2.

Introduction: Resistive breathing (RB) is the hallmark of diseases of airway obstruction, such as chronic obstructive pulmonary disease (COPD), especially during exacerbations. RB results in increased mechanical stress and inflammation in the lung of previously healthy mice. Cigarette smoke (CS) exposure, the main cause of COPD, also induces pulmonary inflammation. Aim: To investigate the potential synergistic effect of the combination of RB with CS exposure model. Methods: Mice were exposed to CS of 5 reference cigarettes, 4 times daily, 5 days per week for one month. Control mice were exposed to room air. By the end of the month mice were subjected to RB for 24 hours by tracheal banding or were sham operated. Tracheal banding was induced by placing the mice under a surgical microscope, the trachea was exposed and a nylon band was placed around the trachea and firmly sutured to provide a surface area 50% of the initial. Following 24 hours, respiratory system mechanics were assessed by the forced oscillation technique. Bronchoalveolar lavage (BAL) was performed and total and differential cell count and total protein level were measured in BAL fluid. Results: CS exposure was associated with increased BAL cellularity (10-fold to ctr, p Conclusions: Combining resistive breathing with cigarette smoke exposure results into augmented inflammatory responses.

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca2+ oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca2+ levels, nor with a significantly changed Ca2+ response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca2+ oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca2+ oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca2+ oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.

Calmodulin (CaM) is a cytoplasmic multifunctional calcium (Ca2+)-binding messenger that interacts with the cardiac ryanodine receptor (RyR2), a large transmembrane Ca2+ channel that mediates Ca2+ release from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Recent genetic studies have reported CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders. Herein, we have investigated the effect of four novel missense CaM mutations, identified in two patients presenting with long QT syndrome (LQTS) (N98I, D134H), and two patients with clinical features of both LQTS and catecholaminergic polymorphic ventricular tachycardia (CPVT), (D132E and Q136P), relative to the biophysical and biochemical properties of wild type CaM (CaMWT). We used CD spectroscopy to examine the thermal stability of CaMWT and mutant proteins. In the absence of Ca2+, thermodynamic values for all proteins were similar. In contrast, in the presence of Ca2+, there was a significant decrease in the stability of the five proteins following the order CaMWT> CaMN98I > CaMD132E > CaMQ136P > CaMD134H. Further Ca2+-binding studies revealed that all CaM mutations significantly reduce the Ca2+-binding affinity of CaMWT. CaMQ136P protein exhibited a ∼7-fold reduced Ca2+-binding affinity compared to CaMWT, while CaMD132E had a ∼14-fold reduction. Furthermore, biochemical analysis revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Our findings confirm our previous observations suggesting that the clinical presentation of LQTS or CPVT associated with these four CaM mutations may involve both altered intrinsic Ca2+-binding as well as dysregulation of RyR2-mediated Ca2+ release via aberrant interaction of CaM with RyR2.

Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation-contraction coupling. Defective CaM-RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM(F90L)) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM(F90L). F90L confers a deleterious effect on protein stability. Ca(2+)-binding studies reveal reduced Ca(2+)-binding affinity and a loss of co-operativity. Moreover, CaM(F90L) displays reduced RyR2 interaction and defective modulation of [(3)H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca(2+) release via aberrant CaM(F90L)-RyR2 interaction is a potential mechanism that underlies familial IVF.