Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

Pitfalls on computing liquid-liquid phase equilibria using the k-value method

Poster presented in the 11th Mediterranean Congress of Chemical Engineering, Barcelona, October 21-24, 2008.

Optimizing the number of stations in arrays measurements: experimental outcomes for different array geometries and the f-k method

Array measurements have become a valuable tool for site response characterization in a non-invasive way. The array design, i.e. size, geometry and number of stations, has a great influence in the quality of the obtained results. From the previous parameters, the number of available stations uses to be the main limitation for the field experiments, because of the economical and logistical constraints that it involves. Sometimes, from the initially planned array layout, carefully designed before the fieldwork campaign, one or more stations do not work properly, modifying the prearranged geometry. Whereas other times, there is not possible to set up the desired array layout, because of the lack of stations. Therefore, for a planned array layout, the number of operative stations and their arrangement in the array become a crucial point in the acquisition stage and subsequently in the dispersion curve estimation. In this paper we carry out an experimental work to analyze which is the minimum number of stations that would provide reliable dispersion curves for three prearranged array configurations (triangular, circular with central station and polygonal geometries). For the optimization study, we analyze together the theoretical array responses and the experimental dispersion curves obtained through the f-k method. In the case of the f-k method, we compare the dispersion curves obtained for the original or prearranged arrays with the ones obtained for the modified arrays, i.e. the dispersion curves obtained when a certain number of stations n is removed, each time, from the original layout of X geophones. The comparison is evaluated by means of a misfit function, which helps us to determine how constrained are the studied geometries by stations removing and which station or combination of stations affect more to the array capability when they are not available. All this information might be crucial to improve future array designs, determining when it is possible to optimize the number of arranged stations without losing the reliability of the obtained results.

Comparative study of RPSALG algorithm for convex semi-infinite programming

The Remez penalty and smoothing algorithm (RPSALG) is a unified framework for penalty and smoothing methods for solving min-max convex semi-infinite programing problems, whose convergence was analyzed in a previous paper of three of the authors. In this paper we consider a partial implementation of RPSALG for solving ordinary convex semi-infinite programming problems. Each iteration of RPSALG involves two types of auxiliary optimization problems: the first one consists of obtaining an approximate solution of some discretized convex problem, while the second one requires to solve a non-convex optimization problem involving the parametric constraints as objective function with the parameter as variable. In this paper we tackle the latter problem with a variant of the cutting angle method called ECAM, a global optimization procedure for solving Lipschitz programming problems. We implement different variants of RPSALG which are compared with the unique publicly available SIP solver, NSIPS, on a battery of test problems.

A two-step mechanism for epigenetic specification of centromere identity and function

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Driver's visibility requirements for roadway delineation. Vol. II: color identification of yellow highway paint as a function of yellow/white pigment mixture ratio. Final report.

Federal Highway Administration, Washington, D.C.

Dynamic, convex, and robust optimization with Bayesian learning for response-guided dosing

Thesis (Ph.D.)--University of Washington, 2016-08

The 1000 brightest hipass galaxies: The H I mass function and Omega(H I)

We present a new, accurate measurement of the H I mass function of galaxies from the HIPASS Bright Galaxy Catalog, a sample of 1000 galaxies with the highest H I peak flux densities in the southern (delta

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy

Objective: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. Methods: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. Results: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (P

Molecular tools for studying puberty in the grey mullet, Mugil cephalus: cloning of cDNAs that regulate reproductive function

The cDNAs coding for the brain GnRHs (AY373449-51), pituitary GH, SL and PRL, and liver IGFs (AY427954-5) were isolated. Partial cDNA sequences of the brain (Cyp19b) and gonadal (Cyp19a) aromatases have also been obtained. These tools would be utilized to study the endocrine regulation of puberty in the grey mullet.

Perceptual and instrumental evaluation of voice and tongue function after carotid endarterectomy

Objective: Laryngeal and tongue function was assessed in 28 patients to evaluate the presence, nature, and resolution of superior recurrent laryngeal and hypoglossal nerve damage resulting from standard open primary carotid endarterectomy (CEA). Methods. The laryngeal and tongue function in 28 patients who underwent CEA were examined prospectively with various physiologic (Aerophone II, laryngograph, tongue transducer), acoustic (Multi-Dimensional Voice Program), and perceptual speech assessments. Measures were obtained from all participants preoperatively, and at 2 weeks and at 3 months postoperatively. Results. The perceptual speech assessment indicated that the vocal quality of roughness was significantly more apparent at the 2-week postoperative assessment than preoperatively. However, by the 3-month postoperative assessment these values had returned to near preoperative levels, with no significant difference detected between preoperative and 3-month postoperative levels or between 2-week and 3-month postoperative levels. Both the instrumental assessments of laryngeal function and the acoustic assessment of vocal quality failed to identify any significant difference on any measure across the three assessment periods. Similarly, no significant impairment in tongue strength, endurance, or rate of repetitive tongue movements was detected at instrumental assessment of tongue function. Conclusions: No permanent changes to vocal or tongue function occurred in this group of participants after primary CEA. The lack of any significant long-term laryngeal or tongue dysfunction in this group suggests that the standard open CEA procedure is not associated with high rates of superior recurrent and hypoglossal nerve dysfunction, as previously believed.

Predicting function from structure: 3D structure studies of the mammalian golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E-rev) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E-rev of nicotine-induced current as a function of extracellular Na+ concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K+/Na+ permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca2+ concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na+, which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.