Capsaicin-induced endocytosis of endogenous presynaptic Ca_{V}2.2 in DRG-spinal cord co-cultures inhibits presynaptic function

The N-type calcium channel, CaV2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their post-synaptic targets in the spinal cord. In this study we have utilized CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. CaV2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in CaV2.2_HA expression and increased association with the post-synaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca2+ influx. The mechanism of this down-regulation of CaV2.2_HA involves a Rab 11a-dependent trafficking process, since dominant-negative Rab11a(S25N) occludes the effect of capsaicin on presynaptic CaV2.2_HA expression, and also prevents the effect of capsaicin on action potential induced Ca2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway

HST Survey of Clusters in Nearby Galaxies. II. Statistical Analysis of Cluster Populations

We present a statistical system that can be used in the study of cluster populations. The basis of our approach is the construction of synthetic cluster color-magnitude-radius diagrams (CMRDs), which we compare with the observed data using a maximum likelihood calculation. This approach permits a relatively easy incorporation of incompleteness (a function of not only magnitude and color, but also radius), photometry errors and biases, and a variety of other complex effects into the calculation, instead of the more common procedure of attempting to correct for those effects. We then apply this procedure to our NGC 3627 data from Paper I. We find that we are able to successfully model the observed CMRD and constrain a number of parameters of the cluster population. We measure a power law mass function slope of alpha = -1.50 +/- 0.07, and a distribution of core radii centered at r_c = 1.53 +/- 0.15 pc. Although the extinction distribution is less constrained, we measured a value for the mean extinction consistent with that determined in Paper I from the Cepheids.Comment: 21 pages, 3 figures accepted for publication by A

Optical identification of the companion to PSR J1911-5958A, the pulsar binary in the outskirts of NGC 6752

We report on the identification of the optical counterpart of the binary millisecond pulsar PSR J1911-5958A, located in the outskirts of the globular cluster NGC 6752. At the position of the pulsar we find an object with V=22.08, B-V=0.38, U-B=-0.49. The object is blue with respect to the cluster main sequence by 0.8 magnitudes in B-V. We argue that the object is the white dwarf companion of the pulsar. Comparison with white dwarf cooling models shows that this magnitude and colors are consistent with a low-mass white dwarf at the distance of NGC 6752. If associated with NGC 6752, the white dwarf is relatively young, <2 Gyr, which sets constraints on the formation of the binary and its ejection from the core of the globular cluster.Comment: Accepted for publication in A&A Letters (September 1st, 2003

Involvement of CaV2.2 channels and α2δ-1 in homeostatic synaptic plasticity in cultured hippocampal neurons

In the mammalian brain, presynaptic CaV2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV2.2 channels at plastic hippocampal synapses remains to be elucidated. Here, we assess CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV2.2 channels at the presynaptic site. This work focussed further on the role of α2δ-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. We find that α2δ-1-overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous α2δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for α2δ-1 during synaptic plastic adaptation

Automating Risk Assessments of Hazardous Material Shipments for Transportation Routes and Mode Selection

The METEOR project at Idaho National Laboratory (INL) successfully addresses the difficult problem in risk assessment analyses of combining the results from bounding deterministic simulation results with probabilistic (Monte Carlo) risk assessment techniques. This paper describes a software suite designed to perform sensitivity and cost/benefit analyses on selected transportation routes and vehicles to minimize risk associated with the shipment of hazardous materials. METEOR uses Monte Carlo techniques to estimate the probability of an accidental release of a hazardous substance along a proposed transportation route. A METEOR user selects the mode of transportation, origin and destination points, and charts the route using interactive graphics. Inputs to METEOR (many selections built in) include crash rates for the specific aircraft, soil/rock type and population densities over the proposed route, and bounding limits for potential accident types (velocity, temperature, etc.). New vehicle, materials, and location data are added when available. If the risk estimates are unacceptable, the risks associated with alternate transportation modes or routes can be quickly evaluated and compared. Systematic optimizing methods will provide the user with the route and vehicle selection identified with the lowest risk of hazardous material release. The effects of a selected range of potential accidents such as vehicle impact, fire, fuel explosions, excessive containment pressure, flooding, etc. are evaluated primarily using hydrocodes capable of accurately simulating the material response of critical containment components. Bounding conditions that represent credible accidents (i.e; for an impact event, velocity, orientations, and soil conditions) are used as input parameters to the hydrocode models yielding correlation functions relating accident parameters to component damage. The Monte Carlo algorithms use random number generators to make selections at the various decision points such as; crash, location, etc. For each pass through the routines, when a crash is randomly selected, crash parameters are then used to determine if failure has occurred using either external look up tables, correlations functions from deterministic calculations, or built in data libraries. The effectiveness of the software was recently demonstrated in safety analyses of the transportation of radioisotope systems for the US Dept. of Energy. These methods are readily adaptable to estimating risks associated with a variety of hazardous shipments such as spent nuclear fuel, explosives, and chemicals

Nerve injury increases native CaV2.2 trafficking in dorsal root ganglion mechanoreceptors

Neuronal N-type (CaV2.2) voltage-gated calcium channels are essential for neurotransmission from primary afferent terminals in the dorsal horn. In this study we have utilized a knock-in mouse expressing CaV2.2 with an inserted extracellular hemagglutinin-tag (CaV2.2_HA), to visualise the distribution of endogenous CaV2.2 in dorsal root ganglion (DRG) neurons and their primary afferents in the dorsal horn. We examined the effect of partial sciatic nerve ligation (PSNL) and found an increase in CaV2.2_HA only in large and medium dorsal root ganglion neurons, and also in deep dorsal-horn synaptic terminals. Furthermore, there is a parallel increase in co-expression with GFRα1, present in a population of low threshold mechanoreceptors, both in large DRG neurons and in their terminals. The increased expression of CaV2.2_HA in these DRG neurons and their terminals is dependent on the presence of the auxiliary subunit α2δ-1, which is required for channel trafficking to the cell surface and to synaptic terminals, and likely contributes to enhanced synaptic transmission at these synapses following PSNL. In contrast the increase of GFRα1 is not altered in α2δ-1 knockout mice. We also found following PSNL there is patchy loss of glomerular synapses immunoreactive for CaV2.2_HA and CGRP or IB4, restricted to the superficial layers of the dorsal horn. This reduction is not dependent on α2δ-1, and likely reflects partial deafferentation of C-nociceptor presynaptic terminals. Therefore, we can distinguish in this pain model two different events affecting specific DRG terminals, with opposite consequences for CaV2.2_HA expression and function in the dorsal horn

The Star Formation History of LGS 3

We have determined the distance and star formation history of the Local Group dwarf galaxy LGS 3 from deep Hubble Space Telescope WFPC2 observations. LGS 3 is intriguing because ground-based observations showed that, while its stellar population is dominated by old, metal-poor stars, there is a handful of young, blue stars. Also, the presence of HI gas makes this a possible ``transition object'' between dwarf spheroidal and dwarf irregular galaxies. The HST data are deep enough to detect the horizontal branch and young main sequence for the first time. A new distance of D=620+/-20 kpc has been measured from the positions of the TRGB, the red clump, and the horizontal branch. The mean metallicity of the stars older than 8 Gyr is Fe/H = -1.5 +/- 0.3. The most recent generation of stars has Fe/H ~ -1. For the first few Gyr the global star formation rate was several times higher than the historical average and has been fairly constant since then. However, we do see significant changes in stellar populations and star formation history with radial position in the galaxy. Most of the young stars are found in the central 63 pc (21''), where the star formation rate has been relatively constant, while the outer parts have had a declining star formation rate.Comment: To appear in The Astrophysical Journal, 26 pages, 14 figures, uses AASTe

Bioengineering of Antibody Fragments: Challenges and Opportunities.

Antibody fragments are used in the clinic as important therapeutic proteins for treatment of indications where better tissue penetration and less immunogenic molecules are needed. Several expression platforms have been employed for the production of these recombinant proteins, from which E. coli and CHO cell-based systems have emerged as the most promising hosts for higher expression. Because antibody fragments such as Fabs and scFvs are smaller than traditional antibody structures and do not require specific patterns of glycosylation decoration for therapeutic efficacy, it is possible to express them in systems with reduced post-translational modification capacity and high expression yield, for example, in plant and insect cell-based systems. In this review, we describe different bioengineering technologies along with their opportunities and difficulties to manufacture antibody fragments with consideration of stability, efficacy and safety for humans. There is still potential for a new production technology with a view of being simple, fast and cost-effective while maintaining the stability and efficacy of biotherapeutic fragments