Novel Gain-of-Function Mutation in the Kv11.1 Channel Found in the Patient with Brugada Syndrome and Mild QTc Shortening.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter IKr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on IKr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the IKr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

Characterization of hERG K+ channel inhibition by the new class III antiarrhythmic drug cavutilide.

Cavutilide (niferidil, refralon) is a new class III antiarrhythmic drug which effectively terminates persistent atrial fibrillation (AF; 84.6% of patients, mean AF duration 3 months) and demonstrates low risk of torsade de pointes (1.7%). ERG channels of rapid delayed rectifier current(IKr) are the primary target of cavutilide, but the particular reasons of higher effectiveness and lower proarrhythmic risk in comparison with other class III IKr blockers are unclear. The inhibition of hERG channels expressed in CHO-K1 cells by cavutilide was studied using whole-cell patch-clamp. The present study demonstrates high sensitivity of IhERG expressed in CHO-K1 cells to cavutilide (IC50 = 12.8 nM). Similarly to methanesulfonanilide class III agents, but unlike amiodarone and related drugs, cavutilide does not bind to hERG channels in their resting state. However, in contrast to dofetilide, cavutilide binds not only to opened, but also to inactivated channels. Moreover, at positive constantly set membrane potential (+ 60 mV) inhibition of IhERG by 100 nM cavutilide develops faster than at 0 mV and, especially, - 30 mV (τ of inhibition was 78.8, 103, and 153 ms, respectively). Thereby, cavutilide produces IhERG inhibition only when the cell is depolarized. During the same period of time, cavutilide produces greater block of IhERG when the cell is depolarized with 2 Hz frequency, if compared to 0.2 Hz. We suggest that, during the limited time after injection, cavutilide produces stronger inhibition of IKr in fibrillating atrium than in non-fibrillating ventricle. This leads to beneficial combination of antiarrhythmic effectiveness and low proarrhythmicity of cavutilide.

Disruption of a Conservative Motif in the C-Terminal Loop of the KCNQ1 Channel Causes LQT Syndrome.

We identified a single nucleotide variation (SNV) (c.1264A > G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein that has never been mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1, using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions in the presence of auxiliary subunit KCNE1 results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect; the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A−B linker of Kv7.1 that was not shown before to be crucial for channel functioning.

Study of Dosage-Dependent Effects of Cytostatic Drugs Using a Fibroblast Cell Culture of the Human Nasal Mucosa

Abstract Introduction Knowing a concentration at which cytostatic drugs are toxic for the nasal fibroblasts will enable the use cytostatic drugs in the clinical practice to prevent excessive cicatrization. Objective To determine the cytostatic concentrations of mitomycin С, doxorubicin, and 5-fluorouracil affecting nasal mucosa fibroblasts. Methods We obtained material during an endonasal dacryocystorhinostomy with the patient's informed consent. The cells were cultivated. Second- to fourth-passage cells were used in the experiments. The cells were stained for vimentin and cluster of differentiation 90 (CD90). An MTS test 3 (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium); cell viability test was performed. Results The cytostatic drugs have a toxic effect on cultivated fibroblasts of the nasal mucosa. This effect is dose-dependent. In terms of reducing the level of tissue fibrotisation in the nasal cavity, the most justified approach is to carry out an experimental study of the effect of mitomycin C, doxorubicin, and 5-fluorouracil at the concentrations of 0.25mg/ml, 0.25mg/ml, and 12.5mg/ml respectively. Conclusion The authors argue that it is inappropriate to use these cytostatic drugs to conduct studies with the goal of analyzing their antifibrotic effect on the nasal mucosa at concentrations that are either lower or higher than the aforementioned ones.

Study of Dosage-Dependent Effects of Cytostatic Drugs Using a Fibroblast Cell Culture of the Human Nasal Mucosa.

Introduction Knowing a concentration at which cytostatic drugs are toxic for the nasal fibroblasts will enable the use cytostatic drugs in the clinical practice to prevent excessive cicatrization. Objective To determine the cytostatic concentrations of mitomycin С, doxorubicin, and 5-fluorouracil affecting nasal mucosa fibroblasts. Methods We obtained material during an endonasal dacryocystorhinostomy with the patient's informed consent. The cells were cultivated. Second- to fourth-passage cells were used in the experiments. The cells were stained for vimentin and cluster of differentiation 90 (CD90). An MTS test 3 (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); cell viability test was performed. Results The cytostatic drugs have a toxic effect on cultivated fibroblasts of the nasal mucosa. This effect is dose-dependent. In terms of reducing the level of tissue fibrotisation in the nasal cavity, the most justified approach is to carry out an experimental study of the effect of mitomycin C, doxorubicin, and 5-fluorouracil at the concentrations of 0.25 mg/ml, 0.25 mg/ml, and 12.5 mg/ml respectively. Conclusion The authors argue that it is inappropriate to use these cytostatic drugs to conduct studies with the goal of analyzing their antifibrotic effect on the nasal mucosa at concentrations that are either lower or higher than the aforementioned ones.

Extracellular membrane vesicles and phytopathogenicity of Acholeplasma laidlawii PG8.

For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria--invasivity, infectivity--and toxigenicity--and to favor to bacterial phytopathogenicity.

Acholeplasma laidlawii PG8 culture adapted to unfavorable growth conditions shows an expressed phytopathogenicity.

Mycoplasmas are the smallest, self-replicating, prokaryotic organisms with avid biochemical potential and spreading in higher eukaryotes in nature. In this study, Acholeplasma laidlawii PG8 cells were cultivated on a deficient medium for 480 days resulting in a mycoplasma culture that was adapted in vitro to unfavorable growth conditions. Cells that survive this condition had decreased sizes (about 0.2 microm) and increased phytopathogenicity. This resulted in more frequent appearance of various morphological alterations when plants of vinca (Vinca minor L.) were infected by adapted mycoplasma cells. The increasing pathogenicity was accompanied by changes in genome expression in these adapted cells. Further studies are needed to explore the exact mechanisms that permit adaptation to unfavorable growth conditions and changes in phytopathogenic potential.