Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers

Spassova M, Tsoneva I, Petrov AG, Petkova JI, Neumann E. Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers. Biophysical Chemistry. 1994;52(3):267-274.Planar lipid bilayers formed from monolayers of diphytanoyl lecithin (DPhL) were found to interact with plasmid DNA (5.6 kbp; M(r) = 3.7 X 10(6)) leading to an increase in the conductance of the membrane. The association of DNA with a lipid bilayer greatly facilitates the transport of the small ions of the main salt KCl. The appearance of long-lived current levels, for instance, of 27.6 pA at V-m = +60 mV membrane voltage, where the actual contact (adsorption) is electrophoretically enhanced, suggests a locally conductive DNA/lipid interaction zone where parts of the DNA strand may be transiently inserted in the bilayer, leaving other parts of the DNA probably protruding out from the outer surface of the bilayer. At V-m = -60 mV, where DNA can be electrophoretically moved away from the membrane, the membrane current is practically zero. This current asymmetry is initially also observed at higher voltages, for instance at 200 mV. However, if the voltage sign (V-m = +200 mV) is changed after a transient positive current (approximate to 15 pA) was observed, there is also now (at V-m = -200 mV) a finite negative current at the negative membrane voltage. Thus, it appears that at V-m = +200 mV the adsorbed parts of the polyelectrolyte DNA are not only transiently inserted in, but actually also electrophoretically pulled through, the porous zones onto the other membrane side leaving the bilayer structure basically intact. These data provide direct electric evidence for the electrophoretic transport of a highly charged and hydrated macromolecule, probably together with the associated gegen-ions, through the thin hydrophobic film of the lipid bilayer

Pesticide Exposure Alters Follicle-Stimulating Hormone Levels in Mexican Agricultural Workers

Organophosphorous pesticides (OPs) are suspected of altering reproductive function by reducing brain acetylcholinesterase activity and monoamine levels, thus impairing hypothalamic and/or pituitary endocrine functions and gonadal processes. Our objective was to evaluate in a longitudinal study the association between OP exposure and serum levels of pituitary and sex hormones. Urinary OP metabolite levels were measured by gas–liquid chromatography, and serum pituitary and sex hormone levels by enzymatic immunoassay and radioimmunoassay in 64 men. A total of 147 urine and blood samples were analyzed for each parameter. More than 80% of the participants had at least one OP metabolite in their urine samples. The most frequent metabolite found was diethylthiophosphate (DETP; 55%), followed by diethylphosphate (DEP; 46%), dimethylthiophosphate (DMTP; 32%), and dimethyldithiophosphate (DMDTP; 31%). However, the metabolites detected at higher concentrations were DMTP, DEP, DMDTP, and dimethylphosphate. There was a high proportion of individuals with follicle-stimulating hormone (FSH) concentrations outside the range of normality (48%). The average FSH serum levels were higher during the heavy pesticide spraying season. However, a multivariate analysis of data collected in all periods showed that serum FSH levels were negatively associated with urinary concentrations of both DMTP and DMDTP, whereas luteinizing hormone (LH) was negatively associated with DMTP. We observed no significant associations between estradiol or testosterone serum levels with OP metabolites. The hormonal disruption in agricultural workers presented here, together with results from experimental animal studies, suggests that OP exposure disrupts the hypothalamic–pituitary endocrine function and also indicates that FSH and LH are the hormones most affected

Reactions of a Be-10 beam on proton and deuteron targets

The extraction of detailed nuclear structure information from transfer reactions requires reliable, well-normalized data as well as optical potentials and a theoretical framework demonstrated to work well in the relevant mass and beam energy ranges. It is rare that the theoretical ingredients can be tested well for exotic nuclei owing to the paucity of data. The halo nucleus Be-11 has been examined through the 10Be(d,p) reaction in inverse kinematics at equivalent deuteron energies of 12,15,18, and 21.4 MeV. Elastic scattering of Be-10 on protons was used to select optical potentials for the analysis of the transfer data. Additionally, data from the elastic and inelastic scattering of Be-10 on deuterons was used to fit optical potentials at the four measured energies. Transfers to the two bound states and the first resonance in Be-11 were analyzed using the Finite Range ADiabatic Wave Approximation (FR-ADWA). Consistent values of the spectroscopic factor of both the ground and first excited states were extracted from the four measurements, with average values of 0.71(5) and 0.62(4) respectively. The calculations for transfer to the first resonance were found to be sensitive to the size of the energy bin used and therefore could not be used to extract a spectroscopic factor.Comment: 16 Pages, 10 figure

Developmental Acquisition of a Rapid Calcium-Regulated Vesicle Supply Allows Sustained High Rates of Exocytosis in Auditory Hair Cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane

Direct neutron capture cross section on Ge 80 and probing shape coexistence in neutron-rich nuclei

Results are presented from the first neutron-transfer measurement on Ge80 using an exotic beam from the Holifield Radioactive Ion Beam Facility at Oak Ridge National Laboratory. Newly measured spins and spectroscopic factors of low-lying states of Ge81 are determined, and the neutron capture cross section on Ge80 was calculated in a direct-semidirect model to provide a more realistic (n,γ) reaction rate for r-process simulations. Furthermore, a region of shape coexistence around N≈50 is confirmed and implications for the magic nature of Ni78 are discussed

Halo nucleus be11: A spectroscopic study via neutron transfer

The best examples of halo nuclei, exotic systems with a diffuse nuclear cloud surrounding a tightly bound core, are found in the light, neutron-rich region, where the halo neutrons experience only weak binding and a weak, or no, potential barrier. Modern direct-reaction measurement techniques provide powerful probes of the structure of exotic nuclei. Despite more than four decades of these studies on the benchmark one-neutron halo nucleus Be11, the spectroscopic factors for the two bound states remain poorly constrained. In the present work, the Be10(d,p) reaction has been used in inverse kinematics at four beam energies to study the structure of Be11. The spectroscopic factors extracted using the adiabatic model were found to be consistent across the four measurements and were largely insensitive to the optical potential used. The extracted spectroscopic factor for a neutron in an nâ., j=2s 1/2 state coupled to the ground state of Be10 is 0.71(5). For the first excited state at 0.32Â MeV, a spectroscopic factor of 0.62(4) is found for the halo neutron in a 1p 1/2 state. © 2012 American Physical Society

Bimodality and Gaps on Globular Cluster Horizontal Branches. II. The Cases of NGC 6229, NGC 1851 and NGC 2808

The outer-halo globular cluster NGC 6229 has a peculiar horizontal-branch (HB) morphology, with clear indications of a bimodal HB and a ``gap" on the blue HB. In this paper, we present extensive synthetic HB simulations to determine whether peculiar distributions in the underlying physical parameters are needed to explain the observed HB morphology. We find that a unimodal mass distribution along the HB can satisfactorily account for the observed HB bimodality, *provided* the mass dispersion is substantially larger than usually inferred for the Galactic globular clusters. In this case, NGC 6229 should have a well-populated, extended blue tail. A truly bimodal distribution in HB masses can also satisfactorily account for the observed HB morphology, although in this case the existence of an extended blue tail is not necessarily implied. The other two well-known bimodal-HB clusters, NGC 1851 and NGC 2808, are briefly analyzed. While the HB morphology of NGC 1851 can also be reproduced with a unimodal mass distribution assuming a large mass dispersion, the same is not true of NGC 2808, for which a bimodal, and possibly multimodal, mass distribution seems definitely required. The problem of gaps on the blue HB is also discussed. Applying the standard Hawarden (1971) and Newell (1973) chi-squared test, we find that the NGC 6229 gap is significant at the 99.7% level. However, in a set of 1,000 simulations, blue-HB gaps comparable to the observed one are present in ~ 6 - 9% of all cases. We employ a new and simple formalism, based on the binomial distribution, to explain the origin of this discrepancy, and conclude that Hawarden's method, in general, substantially overestimates the statistical significance of gaps.Comment: 50 pages (includes 5 tables and 18 multi-panel figures). Higher-resolution versions of Figs. 15a and 15b are available from the first author upon request. To appear in The Astrophysical Journa

Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function