Ion binding in the Open HCN Pacemaker Channel Pore: Fast Mechanisms to Shape “Slow” Channels

IH pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at ∼−34 mV, reflecting a 4:1 selectivity for K over Na. However, IH channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of IH) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10–20 μs or faster) is close to the ion transit time when the pore is occupied by K alone (∼0.5–3 μs), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which “slow” pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials

cAMP Control of HCN2 Channel Mg2+ Block Reveals Loose Coupling between the Cyclic Nucleotide-Gating Ring and the Pore

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile “slow” channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales

Modern approaches to the treatment of genital prolapse in obese women

Introduction. The problem of female genital prolapse (GP) remains in the sportlight of gynecologists, because despite the variety of surgical methods, there are still recurrences of the disease, which are associated not only with the failure of the restored ligaments, fascia, muscles, damaged pelvic floor and perineum, but with the imperfection of the operation. The solution of this problem is especially important in the treatment of patients with extragenital pathology, in particular obesity. The purpose: to optimize the treatment of genital prolapse in obese patients by determining an individual approach to planning surgical treatment taking into account the degree of obesity and concomitant pathology. Materials and methods. We examined 65 patients of which 20 had genital prolapse and obesity (main group), 25 had genital prolapse and normal weight (comparison group), 20 women did not have gynecological diseases and extragenital pathology made up control group. To diagnose obesity and determine its degree we calculated body mass index (BMI). To determine the degree of GP its quantitative assessment was used (POP-Q; 1996). Surgical intervention included transvaginal extirpation of the uterus without appendages, anterior colporrhaphy, colpoperineoraphy with levatoplasty, sacrospinal colpopexy. Transabdominal and laparoscopic colposacropexy in obese women were not used due to the presence of relative contraindications for laparoscopy (cardiovascular disease, respiratory pathology, adhesions, the condition after hernias’ surgery). Therefore, all operations on women with GP and obesity were performed transvaginally due to the inability to perform abdominal access. In comparison group transvaginal surgery was performed. All the groups under study were representative. Before the use of polypropylene mesh "Polymesh" to minimize purulent-septic complications associated with the use of synthetic prostheses aquadissection was performed with 0.9% saline with the addition of 1 g of ceftriaxone per 200 ml. After the operation, the women used suppositories with hyaluronic acid (revitax). Results. The results of surgical treatment have been analyzed and the following data were obtained: recurrences in the main and in the comparison group were 4% (2 women in whom operations were performed with the use of their own tissues without  mesh prosthesis). Infectious complications, dyspareunia and pelvic pain were not observed. Conclusions. Surgical treatment of GP in obese women by using polypropylene mesh "Polymesh" for colposacropexy after transvaginal uterine extirpation increases the effectiveness of treatment  and redduces the number of recurrences. Hydropreparation of the mesh with an antibacterial agent and postoperative use of hyaluronidase intravaginally helps to reduce purulent-septic complications of surgery and improves the patients’quality of life

Investigation of two-photon 2s -> 1s decay in one-electron and one-muon ions

We have studied the radiative decay of the 2s state of one-electron and one-muon ions, where the two-photon mechanism plays an important role. Due to the nuclear size corrections the radiative decay of the 2s state in the electron and muon ions is qualitatively different. Based on the accurate relativistic calculation, we introduced a two-parameter approximation, which makes it possible to describe the two-photon angular-differential transition probability for the polarized emitted photons with high accuracy. The emission of photons with linear and circular polarizations was studied separately. We also investigated the transition probabilities for the polarized initial and final states. The investigation was performed for ions with atomic numbers 1 < Z < 120.Comment: 18 pages, 8 figure

ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations

Investigation on the role of red fox in tuberculosis maintenance community ¿ second opus: experimental infection with a virulent field Mycobacterium bovis strain

Trabajo presentado al: 69th Wildlife Disease Association and 14th European Wildlife Disease Association Conference. Cuenca, Spain. p. 135. 31 agosto-2 septiembre

Pattern Recognition in Pulmonary Tuberculosis Defined by High Content Peptide Microarray Chip Analysis Representing 61 Proteins from M. tuberculosis

Background: Serum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M.tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations. Method: We constructed a high-content peptide microarray with 61 M.tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/2. Findings: Three distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB2, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB2 does no

Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB