NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V_1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V_1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.     

A Potent and Selective Peptide Blocker of the Kv1.3 Channel: Prediction from Free-Energy Simulations and Experimental Confirmation

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.     

Soluble ST2 and Interleukin-33 Levels in Coronary Artery Disease: Relation to Disease Activity and Adverse Outcome

Objectives

ST2 is a receptor for interleukin (IL)-33. We investigated an association of soluble ST2 (sST2) and IL-33 serum levels with different clinical stages of coronary artery disease. We assessed the predictive value of sST2 and IL-33 in patients with stable angina, non-ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI).

Methods

We included 373 patients of whom 178 had stable angina, 97 had NSTEMI, and 98 had STEMI. Patients were followed for a mean of 43 months. The control group consisted of 65 individuals without significant stenosis on coronary angiography. Serum levels of sST2 and IL-33 were measured by ELISAs.

Results

sST2 levels were significantly increased in patients with STEMI as compared to patients with NSTEMI and stable angina as well as with controls. IL-33 levels did not differ between the four groups. During follow-up, 37 (10%) patients died and the combined endpoint (all cause death, MI and rehospitalisation for cardiac causes) occurred in 66 (17.6%) patients. sST2 serum levels significantly predicted mortality in the total cohort. When patients were stratified according to their clinical presentation, the highest quintile of sST2 significantly predicted mortality in patients with STEMI, but not with NSTEMI or stable coronary artery disease. sST2 was a significant predictor for the combined endpoint in STEMI patients and in patients with stable angina. Serum levels of IL-33 were not associated with clinical outcome in the total cohort, but the highest quintile of IL-33 predicted mortality in patients with STEMI.

Conclusions

Serum levels of sST2 are increased in patients with acute coronary syndromes as compared to levels in patients with stable coronary artery disease and in individuals without coronary artery disease. sST2 and IL-33 were associated with mortality in patients with STEMI but not in patients with NSTEMI or stable angina.     

Mast cell stabilizator and antioxidant effects of epidermal growth factor (EGF) on gastric mucosal injury induced by ethanol in rats

The role of epidermal growth factor (EGF), a polypeptide containing 53 amino acids, on protection and repair of ethanol-induced gastric mucosal injury was investigated in rats. In addition, the effects of EGF on the gastric damage were evaluated histopathologically. We used 48 Spraque-Dawley rats which were divided into [corrected] three groups as control rats, ethanol treated rats and ethanol+EGF treated rats. The ethanol group was given a gastric gavage containing 1 ml of 80% ethanol (v/v) prepared in distilled water. EGF (100 microg/kg) was given by intragastric gavage 30 min before the administration of ethanol. We studied histopathological evaluation and the histochemical heterogeneity of mast cells and its degree of degranulation. Besides, gastric tissue malondialdehyde (MDA), protein sulfhydryl groups (SH), and protein carbonyl levels were measured. EGF treatment stabilized mast cells degranulation and had lower polymorphonuclear leukocytes (PMNL) infiltration, ulcer index, histamine, and MDA; protein carbonyl levels were also lower, compared to the non-treated animals. EGF exerts a protective effect on gastric mucosa to ethanol-induced gastric injury probably through antioxidant and mast cell stabilizing mechanism.     

The effect of glutamine on radiation-induced organ damage

Radiation enteritis is a significant clinical problem in patients receiving ionizing radiation directed to the abdomen or pelvis. Although radiation is aimed to be directed against the malignant tissue, adjacent healthy tissues are also affected. The small intestine is the most sensitive organ to radiation. The present study was undertaken to investigate the possible protective effect of glutamine against radiation-induced intestinal, hepatic and pancreatic toxicity. Rats received 1 g/kg/day glutamine for seven days before irradiation and continued for three days after irradiation until sacrifice. Then intestinal, pancreatic and hepatic myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels and caspase-3 activities of the sacrificed rats were measured. Irradiation significantly increased the intestinal and pancreatic MPO and caspase-3 activities and MDA levels in comparison to sham group. Glutamine treatment significantly decreased this elevation. Histopathological examination revealed that the intestinal mucosal structure was preserved and pancreatic inflammation decreased in the glutamine treated group. In irradiation group, NF-kappaB over expression was detected. There was no significant difference in histopathological and biochemical examinations of the liver between the groups. In conclusion, glutamine has beneficial effects on intestinal and pancreatic damage in abdominal irradiation through the inflammatory process and apoptosis.     

Plasma microRNA profiling of children with idiopathic dilated cardiomyopathy

Context: Dilated cardiomyopathy (DCM) is the most common cardiomyopathy in children. MicroRNAs (miRNA) are small RNAs which have regulatory functions in many biological processes.

Objective: We aimed to determine miRNA expression levels in plasma of children with DCM.

Materials and methods: Plasma expression levels of 379 miRNAs were compared between 23 DCM and 26 healthy children.

Results: The expression levels of miR-618, miR-875-3p, miR-205, miR-194, miR-302a, miR-147, and miR-544 were found decreased. The expression levels of miR-518f and miR-454 were found increased in DCM patients.

Discussion: miRNA level differences may provide the chance of using these miRNAs as new biomarkers.     

Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint

During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6]. Here we use dynein-antibody microinjection and the RNAi of ZW10 to explore the role of KD in chromosome behavior during mitosis in mammals. We find that depleting or inhibiting KD prevents the rapid poleward motion of attaching kinetochores but not kinetochore fiber (K fiber) formation. However, after kinetochores attach to the spindle, KD is required for stabilizing kinetochore MTs, which it probably does by generating tension on the kinetochore, and in its absence, chromosome congression is defective. Finally, depleting KD reduces the velocity of anaphase chromosome motion by approximately 40%, without affecting the rate of poleward MT flux. Thus, in addition to its role in silencing the SAC, KD is important for forming and stabilizing K fibers and in powering chromosome motion.     

Evaluating the diagnostic significance of nuclear grooves in thyroid fine needle aspirates with a semiquantitative approach

OBJECTIVE: To clarify the diagnostic significance of nuclear grooves in thyroid fine needle aspirates, to search for other associated nuclear features and to identify possible diagnostic pitfalls. STUDY DESIGN: In total, 67 fine needle aspiration cases were retrospectively reviewed for selected nuclear features, including grooves, intranuclear cytoplasmic invaginations, enlargement, pleomorphism and status of nucleoli. A semiquantitative method was used to estimate the percentage of nuclear grooves in 5 high-power fields with the most frequent nuclear grooves. RESULTS: Unequivocal intranuclear cytoplasmic invaginations were a specific diagnostic feature of papillary carcinoma. In the absence of intranuclear cytoplasmic invaginations, the presence of > or = 20% nuclear grooves was virtually diagnostic of thyroid neoplasms, predominantly papillary carcinoma. Less than 10% of nuclear grooves virtually eliminated the possibility of papillary carcinoma. However, 10-19% of nuclear grooves may be seen in papillary carcinoma and benign lesions; therefore, such cases fell into the diagnostic category of "atypical cytology" or "indeterminate" cytology. Nuclear enlargement with elongation was a readily identifiable feature usually associated with nuclear grooves. Significant pleomorphism was more commonly seen in papillary carcinoma than in benign lesions. CONCLUSION: A semiquantitative method is helpful when nuclear grooves are encountered in thyroid cytology in the absence of other diagnostic features. It can significantly improve the diagnostic specificity of nuclear grooves for papillary carcinoma.     

Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF(+)ClO(4)(-) film was coated on a Pt foil at +0.70 V by electrooxidation of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF(+)HRP(-) was prepared by anion-exchange in a solution of HRP(-) in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy was used to identify PVF, PVF(+)ClO(4)(-), and PVF(+)HRP(-). The immobilized amount of the enzyme in the film was determined by UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined. Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide.