Subcellular localization of the antidepressant-sensitive norepinephrine transporter

<p>Abstract</p> <p>Background</p> <p>Reuptake of synaptic norepinephrine (NE) via the antidepressant-sensitive NE transporter (NET) supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons.</p> <p>Results</p> <p>We elucidate NET localization in brain and superior cervical ganglion (SCG) neurons, aided by a new NET monoclonal antibody, subcellular immunoisolation techniques and quantitative immunofluorescence approaches. We present evidence that axonal NET extensively colocalizes with syntaxin 1A, and to a limited degree with SCAMP2 and synaptophysin. Intracellular NET in SCG axons and boutons also quantitatively segregates from the vesicular monoamine transporter 2 (VMAT2), findings corroborated by organelle isolation studies. At the surface of SCG boutons, NET resides in both lipid raft and non-lipid raft subdomains and colocalizes with syntaxin 1A.</p> <p>Conclusion</p> <p>Our findings support the hypothesis that SCG NET is segregated prior to transport from the cell body from proteins comprising large dense core vesicles. Once localized to presynaptic boutons, NET does not recycle via VMAT2-positive, small dense core vesicles. Finally, once NET reaches presynaptic plasma membranes, the transporter localizes to syntaxin 1A-rich plasma membrane domains, with a portion found in cholera toxin-demarcated lipid rafts. Our findings indicate that activity-dependent insertion of NET into the SCG plasma membrane derives from vesicles distinct from those that deliver NE. Moreover, NET is localized in presynaptic membranes in a manner that can take advantage of regulatory processes targeting lipid raft subdomains.</p

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

AbstractThe four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs

Thermal Conversion of Methane to Acetylene

This report describes the experimental demonstration of a process for the direct thermal conversion of methane to acetylene. The process utilizes a thermal plasma heat source to dissociation products react to form a mixture of acetylene and hydrogen. The use of a supersonic expansion of the hot gas is investigated as a method of rapidly cooling (quenching) the product stream to prevent further reaction or thermal decomposition of the acetylene which can lower the overall efficiency of the process

Novel Battery Testing Procedures and Analytical Methodologies for Hybrid Electric Vehicles

The Idaho National Engineering and Environmental Laboratory has developed novel testing procedures and analytical methodologies to assess the performance of batteries for use in hybrid electric vehicles. Tests include both characterization and cycle life and/or calendar life. Tests have been designed for both Power Assist and Dual Mode applications. Analytical procedures include a battery scaling methodology, the calculation of pulse resistance, pulse power, available energy, and differential capacitance, and the modeling of calendar and cycle life data. At periodic intervals during life testing, a series of Reference Performance Tests are executed to determine changes in the baseline performance of the batteries

cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

<p>Abstract</p> <p>Background</p> <p>The Na⁺/Cl^--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport <it>in vitro </it>and an increased rate of SERT-mediated 5-HT clearance <it>in vivo</it>. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.</p> <p>Results</p> <p>In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT.</p> <p>Conclusion</p> <p>Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.</p

Assessing corrosion in oil refining and petrochemical processing

This paper summarizes the development of an information system used to manage corrosion of metals and alloys by high temperature gases found in many different oil refining, petrochemical, power generation, and chemical processes. The database currently represents about 7.9 million h of exposure time for about 5,500 tests with 89 commercial alloys for a temperature range of 200 – 1,200 °C. The system manages corrosion data from well-defined exposures and determines corrosion product stabilities. New models used in the analysis of thermochemical data for the Fe-Ni-CrCo-C-O-S-N-H system are being compiled. All known phases based upon combinations of the elements have been analyzed to allow complete assessments of corrosion product stabilities. Use of these data allows prediction of stable corrosion products and hence identification of the possible dominant corrosion mechanisms. The system has the potential to be used in corrosion research, alloy development, failure analysis, lifetime prediction, and process operations evaluations. The corrosion mechanisms emphasized are oxidation, sulfidation, sulfidation/oxidation, and carburization