Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1 ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 µM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 µM, 24 hrs) and late-endosomal/lysosomal KIR 2.1 accumulation. Increased KIR 2.1 expression level was also observed in the presence of Nav 1.5 co-expression. Augmented KIR 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 µM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 µM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR 2.1 degradation.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression.

BACKGROUND: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 µM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. METHODS: Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. RESULTS: PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 µM of PA-6 inhibited outward IK1 at -50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 µM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 µM). CONCLUSIONS: 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Success of surgical interventions for direct dental implant-related injuries to the mandibular nerve: a review.

To the best of our knowledge there are no guidelines regarding the surgical management of dental implant-related injuries to the mandibular nerve. This review aims to investigate the success of different surgical interventions. Neurosensory injury to the mandibular branch of the trigeminal nerve can occur during administration of local anaesthetic, elevation of the flap, preparation for osteotomy, and placement of the implant. Surgical interventions include extraction of the implant, external decompression, internal neurolysis, excision of a neuroma, neurorrhaphy, nerve grafting, and low-level laser therapy. The following electronic databases were searched: MEDLINE, EMBASE, and the Cochrane Library. Primary outcome measures included patient-reported outcomes such as pain and altered sensation. A total of 185 publications were obtained, of which 21 were included in the qualitative synthesis (2 randomised controlled trials (RCT), 9 controlled cohort studies, and 10 case reports/series). They were all screened in consideration of the exclusion criteria and appraised using the Cochrane risk of bias tool, the Newcastle Ottawa scale, and the modified Newcastle Ottawa scale. Results were triangulated to evaluate their level of agreement. The extraction of dental implants less than 36hours after injury to the mandibular nerve results in the most successful resolution of neurosensory dysfunction. Various microsurgical techniques have shown less success in obtaining neurosensory recovery than extraction of the implant. However, microsurgery is worthwhile, as it improves neurosensory dysfunction and reduces dysaesthesia in the majority of patients. Direct suturing and external decompression can result in good neurosensory recovery, and nerve grafts are also successful whenever tension-free direct suturing is not possible. Low-level laser therapy has been shown to achieve some neurosensory improvement.

Socket preservation.

Socket preservation maintains bone volume post-extraction in anticipation of an implant placement or fixed partial denture pontic site. This procedure helps compensate for the resorption of the facial bone wall. Socket preservation should be considered when implant placement needs to be delayed for patient or site-related reasons. The ideal healing time before implant placement is six months. Socket preservation can reduce the need for later bone augmentation. By reducing bone resorption and accelerating bone formation it increases implant success and survival. Biomaterials for socket grafting including autograft, allograft, xenograft and alloplast. A bone substitute with a low substitution rate is recommended.

How to measure and predict the molar absorption coefficient of a protein.

The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-326] and is based on data from Edelhoch [1967, Biochemistry 6:1948-1954]). The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon (280), can best be predicted with this equation: epsilon (280) (M-1 cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon (280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon.

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.

Enhanced ventilatory and exercise performance in athletes with slight expiratory resistive loading.

We determined the cardiorespiratory and performance effects of slight (1.5-3.0 cmH2O) expiratory resistive loading (ERL). Twenty-eight highly fit [peak O2 uptake (VO2 peak) = 63.6 +/- 1.3 ml . kg-1 . min-1] athletes (age = 33.5 +/- 1.3 yr) performed paired VO2 peak cycle ergometer tests (control vs. ERL). End-expiratory lung volume was separately determined in a subset of subjects (n = 12) at steady-state 75% maximum power output (POmax) and was found to increase (0.67 +/- 0.29 liter) with ERL. In the VO2 peak tests, peak expiratory pressure at the mouth, mean inspiratory flow, minute ventilation, and O2 pulse were greater with ERL at every intensity level (i.e., 75, 80, 85, and 90% POmax). Increased minute ventilation was largely due to a trend toward increased tidal volume (P < 0.05 at 80% POmax). O2 uptake was greater at 90% POmax with ERL. Increased O2 pulse with ERL at comparative workloads suggests that stroke volume was augmented with ERL. Also, with ERL, athletes attained higher VO2 peak (63.0 +/- 1.4 vs. 60.1 +/- 1.3 ml . kg-1 . min-1) and greater POmax (352.0 +/- 9.9 vs. 345.7 +/- 9.5 W). We conclude that elevated end-expiratory lung volume in response to slight ERL during strenuous exercise served to attenuate both airflow and blood flow limitations, which enhanced exercise capacity.

Music and the brain: the impact of music on an oboist's fight for recovery.

A 20-year-old female, alias Sara, was an aspiring professional oboist who studied music performance at college level. While wading across a river she lost her balance, was swept down river, and suffered profound injuries including severe hypothermia, cardiac arrest, and hypoxic brain injury. While recovering, her family and friends surrounded Sara with music. Her oboe teacher placed Sara's oboe in her hand and played tapes of Sara's past performances. Her mother played recordings of her favourite music in hopes that the music would remind her of her life's passion and, thus, stimulate her mind and soul while she recovered. Two years post-injury, Sara continues to strive to improve her quality of life. In this case study, Sara's musical and medical recovery is detailed using quantitative and qualitative data. This data gathered allows one to provide an analysis of the powerful role of music in Sara's fight to recover.

Activators of phosphorylase kinase alter the cross-linking of its catalytic subunit to the C-terminal one-sixth of its regulatory alpha subunit.

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and delta, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-2-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct heterodimers: a major (alphagamma) and a minor (betadelta) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca2+, and Ca2+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca2+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca2+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca2+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma dimer reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.

The role of the advanced clinical nurse in a hypertension clinic.

The use of advanced clinical nurses (ACNs) has enabled expansion of the Hypertension Clinic at the Johannesburg Hospital. The ACNs care for elderly patients whose hypertension is stable and who require a minimum of drugs. Blood pressure control in this group of patients is satisfactory and the default rate is low. The ACN therefore appears acceptable, at least to the patient. However, problems of continuing education, evaluation and official recognition remain. The case for allowing the ACN to prescribe from a limited pharmacopoeia is presented.