ABSTRACT
α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.
Subject(s)
Prazosin , Receptors, Adrenergic, alpha-1 , Receptors, Adrenergic, alpha-1/metabolism , Tamsulosin , NorepinephrineABSTRACT
Neurotensin (NT) is a 13-residue endogenous peptide found in mammals, with neurotransmission and hormonal roles in the central nervous system and gastrointestinal tract, respectively. The first residue of NT is a pyroglutamate (pGlu) that makes the expression and purification of large amounts of NT with native modification challenging. Here, we describe a simple and efficient procedure for expression and purification of large amounts of NT based on using the small ubiquitin-like modifier (SUMO) as a fusion partner and subsequent enzymatic conversion of the N-terminal glutamine to pGlu. Yields of 13 mg/L and 8 mg/L of pure peptide were obtained from expression in rich and minimal media, respectively. The method is adaptable to expression and purification of proteins and peptides with pGlu modification in a wide range of eukaryotic and prokaryotic expression hosts.
Subject(s)
Neurotensin , Pyrrolidonecarboxylic Acid , Animals , Neurotensin/genetics , Neurotensin/chemistry , Neurotensin/metabolism , Peptides/chemistry , Glutamine , MammalsABSTRACT
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
Subject(s)
Crystallography, X-Ray , Receptors, Adrenergic, alpha-1/chemistry , Binding Sites , HEK293 Cells , Humans , Ligands , Lipids/chemistry , Models, Molecular , Quinazolines/chemistry , Quinazolines/metabolism , Quinoxalines/chemistry , Quinoxalines/metabolism , Receptors, Adrenergic, alpha-2/chemistryABSTRACT
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody-mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell-mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB/c mice were administered various anti-αIIb, anti-ß3 or anti-GPIb antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse-anti-mouse ß3 sera and an anti- αIIb monoclonal antibody (MWReg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.
Subject(s)
Autoantibodies/immunology , Blood Platelets/immunology , Megakaryocytes/pathology , Purpura, Thrombocytopenic, Idiopathic/immunology , Purpura, Thrombocytopenic, Idiopathic/pathology , Animals , Bone Marrow Cells/pathology , Mice , Mice, Inbred BALB CABSTRACT
We investigate the elastic buckling of a triangular prism made of a soft elastomer. A face of the prism is bonded to a stiff slab that imposes an average axial compression. We observe two possible buckling modes which are localized along the free ridge. For ridge angles Ï below a critical value Ï^{â}≈90°, experiments reveal an extended sinusoidal mode, while for Ï above Ï^{â}, we observe a series of creases progressively invading the lateral faces starting from the ridge. A numerical linear stability analysis is set up using the finite-element method and correctly predicts the sinusoidal mode for Ï≤Ï^{â}, as well as the associated critical strain ε_{c}(Ï). The experimental transition at Ï^{â} is found to occur when this critical strain ε_{c}(Ï) attains the value ε_{c}(Ï^{â})=0.44 corresponding to the threshold of the subcritical surface creasing instability. Previous analyses have focused on elastic crease patterns appearing on planar surfaces, where the role of scale invariance has been emphasized; our analysis of the elastic ridge provides a different perspective, and reveals that scale invariance is not a sufficient condition for localization.
ABSTRACT
MAIN CONCLUSION: The propagation of Rafflesia spp. is considered to be important for future development of ornamental and other applications. Thus far, the only successful propagation technique has been grafting. This mini-review succinctly emphasizes what is known about Rafflesia species. Members of the genus Rafflesia (Rafflesiaceae), which are holoparasitic plants known to grow on a host vine, Tetrastigma sp., are widely spread from the Malayan Peninsula to various islands throughout Indonesia. The plant's geographical distribution as well as many other aspects pertaining to the basic biology of this genus have still not been studied. The young flower buds and flowers of wild Rafflesia hasseltii Suringar, Rafflesia keithii Meijer and Rafflesia cantleyi Solms-Laubach are used in local (Malaysia and Indonesia) traditional ethnomedicine as wound-healing agents, but currently no formal published research exists to validate this property. To maintain a balance between its ethnomedicinal and ornamental use, and conservation, Rafflesia spp. must be artificially cultivated to prevent overexploitation. A successful method of vegetative propagation is by host grafting using Rafflesia-impregnated Tetrastigma onto the stem of a normal Tetrastigma plant. Due to difficulties with culture contamination in vitro, callus induction was only accomplished in 2010 for the first time when picloram and 2,4-D were added to a basal Murashige and Skoog medium, and the tissue culture of holoparasitic plants continues to be extremely difficult. Seeds harvested from fertile fruit may serve as a possible method to propagate Rafflesia spp. This paper provides a brief synthesis on what is known about research related to Rafflesia spp. The objective is to further stimulate researchers to examine, through rigorous scientific discovery, the mechanisms underlying the ethnomedicinal properties, the flowering mechanisms, and suitable in vitro regeneration protocols that would allow for the fortification of germplasm conservation.
Subject(s)
Conservation of Natural Resources , Magnoliopsida/growth & development , Flowers/growth & development , Fruit , Germination , Magnoliopsida/physiology , Phylogeny , Reproduction , Seeds , Tissue Culture TechniquesABSTRACT
We investigate how natural curvature affects the configuration of a thin elastic rod suspended under its own weight, as when a single strand of hair hangs under gravity. We combine precision desktop experiments, numerics, and theoretical analysis to explore the equilibrium shapes set by the coupled effects of elasticity, natural curvature, nonlinear geometry, and gravity. A phase diagram is constructed in terms of the control parameters of the system, namely the dimensionless curvature and weight, where we identify three distinct regions: planar curls, localized helices, and global helices. We analyze the stability of planar configurations, and describe the localization of helical patterns for long rods, near their free end. The observed shapes and their associated phase boundaries are then rationalized based on the underlying physical ingredients.
ABSTRACT
We present results from an experimental investigation of the indentation of nonspherical pressurized elastic shells with a positive Gauss curvature. A predictive framework is proposed that rationalizes the dependence of the local rigidity of an indented shell on the curvature in the neighborhood of the locus of indentation, the in-out pressure differential, and the material properties. In our approach, we combine classic theory for spherical shells with recent analytical developments for the pressurized case, and proceed, for the most part, by analogy, guided by our own experiments. By way of example, our results elucidate why an eggshell is significantly stiffer when compressed along its major axis, as compared to doing so along its minor axis. The prominence of geometry in this class of problems points to the relevance and applicability of our findings over a wide range of length scales.
Subject(s)
Egg Shell/chemistry , Models, Theoretical , Animals , Biomechanical Phenomena , Chickens , Elasticity , Linear Models , PressureABSTRACT
The increase in platelets in patients with immune thrombocytopenia (ITP) by intravenous administration of human immunoglobulin concentrates (IVIG) reflects a therapeutic immunomodulatory intervention targeted at the disturbed immune response in many inflammatory and autoimmune disorders. These immunoglobulin concentrates contain large numbers of antibodies as well as trace levels of various other immunologically active molecules. Clinical and laboratory studies have documented various mechanisms of action of IVIG. The complex network of immunological reactions resulting from the infusion of IVIG includes changes in several cytokines, interactions with dendritic cells, T- and B- lymphocyte effects, macrophage effects, mediated by distinct Fc-gamma receptors. In addition, effects on complement components and apoptosis have also been observed. Synergism between the different elements of the immune response characterizes the beneficial effects of IVIG in inflammatory and autoimmune disorders. They have immunopathogeneses and clinical manifestations which are difficult to define and therefore IVIG treatment indications remain heterogeneous. Dose finding studies are missing for most of the indications of the drug. In future research, defining the appropriate subgroups of patients should be undertaken. This may be accomplished by prospective registries collecting data on large numbers of patients with long-term follow-up. Controlled clinical and laboratory studies may follow based on new, validated patient selection criteria and focused on mechanisms of action, leading to more evidence-based indications.
Subject(s)
Autoimmune Diseases/therapy , Immunoglobulins, Intravenous/pharmacology , Immunologic Factors/pharmacology , Autoimmune Diseases/immunology , Cytokines/physiology , Dendritic Cells/immunology , Forecasting , Humans , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Immunoglobulins, Intravenous/therapeutic use , Immunologic Factors/therapeutic use , Inflammation/immunology , Inflammation/therapy , Lymphocyte Subsets/immunology , Macrophages/immunology , Phagocytosis/drug effects , Receptors, IgG/physiologyABSTRACT
Intravenous immunoglobulin (IVIg) is used to treat an ever-increasing number of autoimmune diseases. While the exact mechanism of action of IVIg has remained elusive, many theories have been suggested, including mononuclear phagocytic system blockade, autoantibody neutralization by anti-idiotype antibodies, accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc receptor, cytokine modulation and complement neutralization. More recently, a key role for dendritic cells (DC) in the amelioration of autoimmunity by IVIg has been suggested. Here we will focus on the role that DC may play in IVIg function using data from both mouse and human studies.
Subject(s)
Dendritic Cells/immunology , Immunoglobulins, Intravenous/pharmacology , Animals , Autoimmunity , Humans , Immunoglobulins, Intravenous/therapeutic use , MiceABSTRACT
A study of solar-wind hydrogen and helium temperature observations collected by the Wind spacecraft offers compelling evidence of heating by an Alfvén-cyclotron dissipation mechanism. Observations are sorted by the rate of Coulomb interactions, or collisional age, in the plasma and the differential flow between the two species. We show that helium is preferentially heated perpendicular to the magnetic field direction by more than a factor of 6 when the flow between the species is small relative to the Alfvén wave speed and collisions are infrequent. These signatures are consistent with predictions of dissipation in the presence of multiple ion species. We also report an unexpected result: observations of efficient heating of helium parallel to the magnetic field for large differential flow relative to the sound speed.
ABSTRACT
Hyalomma anatolicum anatolicum nymphs were collected from two localities in the Sudan: Eddamer in Northern Sudan and Wad-Medani in Central Sudan. They were allowed to moult to adult ticks, which were assessed for Theileria infection in their salivary glands using Feulgen stain. At Eddamer, 49.6% of 123 ticks examined were infected with Theileria and the mean intensity of infection was 1.3 (i.e. the number of infected acini/number of infected ticks). At Wad-Medani, 8.6% of 162 ticks were infected and the mean intensity of infection was 7.9. The prevalence of infection was higher in female than in male ticks at both localities. When adult H. a. anatolicum were applied onto two susceptible calves, both animals developed the severe form of theileriosis.
Subject(s)
Theileria annulata/isolation & purification , Theileriasis/epidemiology , Theileriasis/transmission , Ticks/parasitology , Animals , Cattle , Female , Male , Salivary Glands/parasitology , Sex Factors , Sudan/epidemiologyABSTRACT
Cardiac tachyarrhythmias have rarely been studied in young patients with myotonic dystrophy type 1 (DM1). The authors observed major cardiac rhythm disturbances in 11 patients aged 10 to 18 years. Tachyarrhythmic events were more frequent than impulse conduction disorders. Wide variations in CTG expansion were observed among the population. Since physical exercise was a prominent arrhythmogenic factor, systematic exercise tests with EKG monitoring may be indicated in young patients with DM1.
Subject(s)
Arrhythmias, Cardiac/etiology , Myotonic Dystrophy/complications , Adolescent , Adrenergic beta-Antagonists/therapeutic use , Atrial Flutter/etiology , Atrial Flutter/surgery , Catheter Ablation , Child , Chromosomes, Human, Pair 19/genetics , Death, Sudden, Cardiac/etiology , Defibrillators, Implantable , Exercise Test , Female , Heart Arrest/etiology , Humans , Male , Myotonic Dystrophy/diagnosis , Myotonic Dystrophy/genetics , Retrospective Studies , Tachycardia, Ventricular/drug therapy , Tachycardia, Ventricular/etiology , Trinucleotide Repeats , Ventricular Fibrillation/etiology , Ventricular Fibrillation/surgeryABSTRACT
The first case report introducing the concept of cardiac resynchronisation therapy (CRT) was published less than 10 years ago, opening the way to the development of the first successful non-pharmacological treatment of congestive heart failure (CHF). The now routine implantation of CRT systems is applicable to multitudes of patients as adjunctive therapy in advanced CHF. This technique has transformed the traditional concepts associated with stimulation of the heart, and is now applied not only to restore an appropriate heart rate, but also to change the process of cardiac mechanical activation. Since it must be integrated within a comprehensive and multidisciplinary CHF management program, CRT has changed the practice of experts in the field of cardiac pacing. CRT in the management of CHF was ultimately validated in 2 randomised trials. MUSTIC, the first trial, compared in a single-blind, 3x3 months crossover design active versus inactive biventricular stimulation in a group of patients in sinus rhythm and another group in atrial fibrillation. Both phases of the trial were completed by 48 patients, with significant positive effects conferred by CRT on the distance walked in 6 min and on peak oxygen consumption. The number of hospitalizations for management of CHF was decreased by 2/3 (P<0.05), and 85% of patients preferred the atrio-biventricular over the inactive stimulation mode (P<0.001). These results were amply confirmed by the parallel-design MIRACLE trial. The current indications for CRT, diagnostic tools to assist in its implementation, and limitations of this new therapeutic adjunct are further discussed in this review.
Subject(s)
Cardiac Pacing, Artificial , Defibrillators, Implantable , Electric Countershock , Heart Failure/therapy , Clinical Trials as Topic , Heart Failure/physiopathology , HumansABSTRACT
The evaluation of multisite stimulation with a haemodynamic aim has since its origin clashed with the absence of definition of a simple method of identifying candidates and of evaluation of the effects of treatment. In this pilot work, 66 patients were selected on electromechanical criteria obtained from a desynchronisation model identified from simple echographic parameters. The short term results demonstrate important modifications, differing according to the type of patient undergoing implantation. These results reject the basis of a prospective multicentric study aimed at validating the concept of ventricular resynchronisation.
Subject(s)
Arrhythmias, Cardiac/diagnostic imaging , Echocardiography/methods , Heart Diseases/physiopathology , Heart Diseases/surgery , Heart Rate/physiology , Cardiomyopathy, Dilated/diagnostic imaging , Cardiomyopathy, Dilated/physiopathology , Cardiomyopathy, Dilated/surgery , Heart Diseases/diagnostic imaging , Humans , Models, Cardiovascular , Monitoring, Intraoperative/methods , Myocardial Ischemia/diagnostic imaging , Myocardial Ischemia/physiopathology , Myocardial Ischemia/surgery , Ventricular Dysfunction, Left/diagnostic imaging , Ventricular Dysfunction, Left/physiopathology , Ventricular Dysfunction, Left/surgerySubject(s)
CD40 Ligand/physiology , Platelet Aggregation , Animals , Bleeding Time , CD40 Ligand/genetics , Mice , Mice, Knockout , Stress, MechanicalABSTRACT
The interaction between von Willebrand factor (VWF) and glycoprotein (GP) Ib results in platelet agglutination and activation of many signaling intermediates. To determine if VWF-dependent platelet activation requires the participation of pivotal transmembrane signaling pathways, we analyzed VWF-dependent platelet activation profiles following inhibition of several transmembrane signaling intermediates. This was accomplished using porcine VWF, which has been shown to interact with human GPIb independently of shear stress or ristocetin. Platelet alpha (CD62) and lysozomal granule release (CD63), microparticle formation, and platelet agglutination/aggregation were evaluated. The ability of signaling inhibitors to prevent VWF-dependent platelet activation was compared to their ability to inhibit thrombin-dependent activation. The results demonstrate that VWF-dependent platelet activation can occur independently of the activities of protein kinase C (PKC), wortmannin-sensitive phosphatidylinositide 3-kinase, and phospholipase C, as well as independently of elevations in the concentration of intracellular calcium. In sharp contrast, these transmembrane signaling intermediates are required for thrombin-dependent platelet activation. In addition, thrombin-dependent but not VWF-dependent platelet activation was associated with elevations in the concentration of intracellular calcium under the conditions used. The family of signaling intermediates which appeared to be pivotal for both thrombin- and VWF-dependent platelet activation were the protein tyrosine phosphatases and the serine/threonine phosphatases. It is concluded that thrombin-dependent platelet activation relies on the activation of several transmembrane signaling pathways, whereas VWF-dependent platelet activation is dependent upon the activity of protein phosphatases. Inhibition of these phosphatases in vivo may provide a novel therapeutic approach for treating VWF-dependent platelet disorders such as thrombotic thrombocytopenic purpura or arterial thrombosis.
Subject(s)
Platelet Activation/physiology , von Willebrand Factor/physiology , Adenosine Diphosphate/pharmacology , Animals , Antigens, CD/blood , Blood Platelets/drug effects , Blood Platelets/physiology , Calcium/blood , Cell Membrane/physiology , Humans , In Vitro Techniques , P-Selectin/blood , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/blood , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Platelet Aggregation/physiology , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins , Signal Transduction , Species Specificity , Sus scrofa , Tetraspanin 30 , Thrombin/pharmacology , Thrombin/physiology , von Willebrand Factor/pharmacologyABSTRACT
Multisite biventricular pacing therapy offers significant clinical improvement in some stimulated patients with electrocardiographic criteria of cardiac dyssynchrony. However, observational data increasingly suggest that patients suffering from congestive heart failure in presence of modest QRS widening may also derive benefit from cardiac resynchronization therapy (CRT), and that some patients can be significantly improved clinically after system implantation despite no apparent change in QRS width. This pilot study explored the value of an echocardiographic model to identify cardiac electromechanical dyssynchrony parameters (EDP) in candidates for CRT, and their potential correction after implantation. The study included 66 consecutive CRT recipients of CRT in NYHA functional class III or IV who had one or more atrioventricular, interventricular or intraventricular dyssynchrony criteria. An immediate improvement was observed in 85% of the population with a partial or total correction of their EDP. However, the modifications in EDP differed considerably between recipients of de novo CRT systems and patients with previously implanted standard pacing systems upgraded with the implantation of a left ventricular lead. EDP measurements appear to identify potential candidates for CRT, and to confirm the success of system implantation.
Subject(s)
Cardiac Pacing, Artificial , Cardiomyopathy, Dilated/therapy , Echocardiography , Ventricular Dysfunction, Left/therapy , Atrioventricular Node/physiopathology , Cardiac Pacing, Artificial/methods , Cardiomyopathy, Dilated/diagnostic imaging , Cardiomyopathy, Dilated/physiopathology , Electrocardiography , Electrophysiologic Techniques, Cardiac , Humans , Longitudinal Studies , Myocardial Contraction , Pacemaker, Artificial , Pilot Projects , Prospective Studies , Ventricular Dysfunction, Left/diagnostic imagingABSTRACT
Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.