Efficient Photoionization-Loading of Trapped Cadmium Ions with Ultrafast Pulses

Atomic cadmium ions are loaded into radiofrequency ion traps by photoionization of atoms in a cadmium vapor with ultrafast laser pulses. The photoionization is driven through an intermediate atomic resonance with a frequency-quadrupled mode-locked Ti:Sapphire laser that produces pulses of either 100 fsec or 1 psec duration at a central wavelength of 229 nm. The large bandwidth of the pulses photoionizes all velocity classes of the Cd vapor, resulting in high loading efficiencies compared to previous ion trap loading techniques. Measured loading rates are compared with a simple theoretical model, and we conclude that this technique can potentially ionize every atom traversing the laser beam within the trapping volume. This may allow the operation of ion traps with lower levels of background pressures and less trap electrode surface contamination. The technique and laser system reported here should be applicable to loading most laser-cooled ion species.Comment: 11 pages, 12 figure

C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential

-1 Receptor Modulation of Acid-Sensing Ion Channel a (ASIC1a) and ASIC1a-Induced Ca 2ϩ Influx in Rat Cortical Neurons

ABSTRACT Acid-sensing ion channels (ASICs) are proton-gated cation channels found in peripheral and central nervous system neurons. The ASIC1a subtype, which has high Ca 2ϩ permeability, is activated by ischemia-induced acidosis and contributes to the neuronal loss that accompanies ischemic stroke. Our laboratory has shown that activation of receptors depresses ion channel activity and [Ca 2ϩ ] i dysregulation during ischemia, which enhances neuronal survival. Whole-cell patch-clamp electrophysiology and fluorometric Ca 2ϩ imaging were used to determine whether receptors regulate the function of ASIC in cultured rat cortical neurons. Bath application of the selective ASIC1a blocker, psalmotoxin1, decreased proton-evoked [Ca 2ϩ ] i transients and peak membrane currents, suggesting the presence of homomeric ASIC1a channels. The pan-selective -1/-2 receptor agonists, 1,3-di-o-tolyl-guanidine (100 M) and opipramol (10 M), reversibly decreased acid-induced elevations in [Ca 2ϩ ] i and membrane currents. Pharmacological experiments using receptor-subtype-specific agonists demonstrated that -1, but not -2, receptors inhibit ASIC1a-induced Ca 2ϩ elevations. These results were confirmed using the irreversible receptor antagonist metaphit (50 M) and the selective -1 antagonist BD1063 (10 nM), which obtunded the inhibitory effects of the -1 agonist, carbetapentane. Activation of ASIC1a was shown to stimulate downstream Ca 2ϩ influx pathways, specifically N-methyl-D-aspartate and (Ϯ)-␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and voltage-gated Ca 2ϩ channels. These subsequent Ca 2ϩ influxes were also inhibited upon activation of -1 receptors. These findings demonstrate that -1 receptor stimulation inhibits ASIC1a-mediated membrane currents and consequent intracellular Ca 2ϩ accumulation. The ability to control ionic imbalances and Ca 2ϩ dysregulation evoked by ASIC1a activation makes receptors an attractive target for ischemic stroke therapy. Acid-sensing ion channels are a class of ligand-gated channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed in both peripheral and central nervous system neurons . Thus far, four genes (ASIC1-ASIC4) and two splice variants of ASIC1 and ASIC2 (a and b) have been cloned ) that encode protein subunits that form functional proton-gated homomultimeric or heteromultimeric channels . The pH of half-maximal activation and the tissue expression patterns differ between each channel subtype. One of the most common ASIC subtypes in the central nervous system (CNS) contains the ASIC1a subunit, which can form homomultimeric or heteromultimeric channels with ASIC2a . These channels are activated by pH Յ 7 and have a pH of half-maximal activation of ϳ6.

ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart

The full-length cDNAs of two different α-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different β-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart.Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected.Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3–15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 ± 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions.The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 μm 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential.Rilmakalim (1 μm), diazoxide (300 μm) and metabolic inhibition increased the slope conductance measured at −55 mV by a factor of 9.0 (±1.8), 2.5 (±0.2) and 3.9 (±1.7), respectively. The effects were reversed by glibenclamide (1 μm).Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall