Asymptomatic Profound Sinus Bradycardia (Heart Rate ≤45) in Non-small Cell Lung Cancer Patients Treated with Crizotinib

Abstract:Crizotinib, a dual MET/ALK inhibitor, is now in advanced clinical development for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We have observed several patients who developed profound but asymptomatic sinus bradycardia (HR ≤45) during the course of crizotinib treatment. Herein, we describe the clinical characteristics of three separate patients enrolled in the A8081001 trial (NCT00585195) who developed asymptomatic profound sinus bradycardia with their accompanying electrocardiogram tracings

Clinical and biochemical changes in 53 Swedish dogs bitten by the European adder - Vipera berus

<p>Abstract</p> <p>Background</p> <p>Every year many dogs in Sweden are bitten by <it>Vipera berus</it>, the only venomous viper in Sweden. This prospective study investigated clinical signs, some biochemical parameters, treatment, and progress of disease after snakebite in 53 dogs. Effects of treatment with and without glucocorticoids were evaluated.</p> <p>Methods</p> <p>All fifty-three dogs bitten by <it>Vipera berus </it>were examined the same day the dog was bitten and the next day. Two more examinations during 23 days post snake bite were included. Creatinine, creatine kinase (CK), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), alkaline phosphatase (ALP) and bile acid results were followed through 3 to 4 samplings from 34 of the dogs.</p> <p>Results</p> <p>All dogs had variable severity of local swelling in the bite area and 73 per cent had affected mental status. Initial cardiac auscultation examination was normal in all dogs, but six dogs had cardiac abnormalities at their second examination, including cardiac arrhythmias and cardiac murmurs. All dogs received fluid therapy, 36 dogs were given analgesics, 22 dogs were treated with glucocorticoids, and ten dogs were treated with antibiotics. Evidence of transient muscle damage (increased CK) was seen one day after the snake bite in 15 (54%) of 28 sampled dogs. Moderate changes in hepatic test results occurred in 1 dog and several dogs (22 of 34) had transient, minor increases in one or more hepatic test result. No dog died during the observation period as a consequence of the snake bite.</p> <p>Conclusions</p> <p>Snake bite caused local swelling in all dogs and mental depression of short duration in most dogs. Some dogs had transient clinical signs that could be indicative of cardiac injury and some other had transient biochemical signs of liver injury. Treatment with glucocorticoids did not have any clear positive or negative effect on clinical signs and mortality.</p

Effect of Oxidative Stress on Homer Scaffolding Proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress

Ryanodine Receptors in Muscarinic Receptor-mediated Bronchoconstriction

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma

Excitation-Contraction Coupling in Airway Smooth Muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions

Nanomolar potency and selectivity of a Ca2+ release-activated Ca2+ channel inhibitor against store-operated Ca2+ entry and migration of vascular smooth muscle cells

BACKGROUND AND PURPOSE The aim was to advance the understanding of Orai proteins and identify a specific inhibitor of the associated calcium entry mechanism in vascular smooth muscle cells (VSMCs). EXPERIMENTAL APPROACH Proliferating VSMCs were cultured from human saphenous veins. Intracellular calcium was measured using fura‐2, whole‐cell current was recorded using patch‐clamp and cell migration quantified in modified Boyden chambers. Subcellular protein localization was determined by microscopy. Isometric tension was recorded from mouse aortic rings. KEY RESULTS Molecular disruption and rescue experiments indicated the importance of Orai1 in calcium entry caused by store depletion evoked passively or by platelet‐derived growth factor (PDGF), suggesting the presence of Ca2+ release‐activated Ca2+ (CRAC) channels like those of the immune system. The CRAC channel blocker, S66, was a potent inhibitor of the VSMC signals, IC50 26 nM, which was almost two orders of magnitude greater than with leucocytes. S66 had no effect on PDGF‐ and ATP‐evoked calcium release, overexpressed transient receptor potential canonical (TRPC)5 channels, native TRPC1/5‐containing channels, stromal interaction molecule 1 clustering, non‐selective cationic current evoked by store depletion and phenylephrine‐evoked aortic contraction. S66 reduced PDGF‐evoked VSMC migration while having only modest effects on cell proliferation and no effect on cell viability. CONCLUSIONS AND IMPLICATIONS The data suggest that Orai1 has a role in human VSMC migration, and that a CRAC channel inhibitor has high potency and selectivity for the associated calcium entry, suggesting a distinct characteristic of vascular CRAC channels and the potential for selective chemical suppression of vascular remodelling

A novel advanced test system for polymer electrolyte membrane water electrolysis based on hydraulic cell compression

In this work, a novel polymer electrolyte membrane water electrolyzer (PEMWE) test cell based on hydraulic single-cell compression is described. In this test cell, the current density distribution is almost homogeneous over the active cell area due to hydraulic cell clamping. As the hydraulic medium entirely surrounds the active cell components, it is also used to control cell temperature resulting in even temperature distribution. The PEMWE single-cell test system based on hydraulic compression offers a 25 cm2 active surface area (5.0 × 5.0 cm) and can be operated up to 80◦C and 6.0 A/cm2. Construction details and material selection for the designed test cell are given in this document. Furthermore, findings related to pressure distribution analyzed by utilizing a pressure-sensitive foil, the cell performance indicated by polarization curves, and the reproducibility of results are described. Experimental data indicate the applicability of the presented testing device for relevant PEMWE component testing and material analysi