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Silver clusters were assembled in helium droplets of different sizes ranging from 104 to 1011 atoms. The clusters were heated upon laser irradiation at 355 nm, and evaporation dynamics of He atoms were studied by quadrupole mass spectroscopy using signals from He+, He2+, and He4+ splitter ions. We found that for droplets containing less than 107 atoms the laser irradiation leads to evaporation of He atoms. However, the laser irradiation leads to the breakup of the large droplets into smaller ones.

X-raying superfluid helium droplets When physicists rotate the superfluid 4 He, it develops a regular array of tiny whirlpools, called vortices. The same phenomenon should occur in helium droplets half a micrometer in size, but studying individual droplets is tricky. Gomez et al. used x-ray diffraction to deduce the shape of individual rotating droplets and image the resulting vortex patterns, which confirmed the superfluidity of the droplets. They found that superfluid droplets can host a surprising number of vortices and can rotate faster than normal droplets without disintegrating. Science , this issue p. 906

This chapter aims to look at the properties of large helium nanodroplets from two different perspectives: (i) helium droplets as hosts for assembling and studying clusters at low temperatures; and (ii) helium droplets as systems to be studied on their own. First, the thermodynamics and excitations in large droplets are presented, followed by a primer on the rate of droplet cooling in vacuo. The chapter then proceeds with the description on producing and characterizing the droplets. This is followed by a discussion on the kinetics for different cluster aggregation regimes, such as that for single- and multiple-centre aggregation. Then, experiments involving the spectroscopy of foreign particles and the deposition of metallic clusters for electron microscopy studies are described. Finally, results from recent X-ray coherent diffractive imaging experiments with pure and doped helium nanodroplets are summarized.

Lectin-glycoconjugate interactions are essential for numerous cellular and extracellular events. Binding stoichiometry is one of several factors that regulate the initiation and outcome of lectin-mediated biological functions. Therefore, the knowledge of accurate stoichiometry of glycoconjugate binding is pivotal for delineating the molecular mechanism of lectin-dependent processes. Quantitative precipitation and isothermal titration calorimetry can reliably determine the stoichiometry of glycoconjugate recognition by lectins. Stoichiometry plays important roles in affinity enhancement and it influences the structures of lectin-glycoconjugate cross-linked complexes. Correct stoichiometry helped reveal that the binding epitopes (glycans) on multi- and polyvalent glycoconjugates possess decreasing microscopic binding affinities for lectins and the binding of lectin involves an internal diffusion process called “bind and jump.” Interestingly, in certain cases, stoichiometry is unable to regulate affinity enhancement, binding thermodynamics, kinetics of cross-linking, and the lattice structures of the cross-linked complexes.

Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca 2+ channels. After membrane depolarization, Ca 2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca 2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca 2+ channel Ca V 1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with β 1 subunits prominent in brain, as well as β 2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca 2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly 406 . As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at ≈50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism.

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene. ispartof: Neuron vol:55 issue:4 pages:615-632 ispartof: location:United States status: published

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. We investigated the requirements for the different MB neurons, specifically the α/β versus the γ neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. We expressed the rutabaga (rut)-encoded adenylyl cyclase in either the γ or α/β neurons and examined the effects on restoring olfactory associative learning and memory of rut mutant flies. We also expressed a temperature-sensitive shibire (shi) transgene in these neuron sets and examined the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning. Our results indicate that although we did not detect odor-pair-specific learning using GAL4 drivers that primarily express in γ neurons, expression of the transgenes in a subset of α/β neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shits1.

[structure: see text] The viability of proteins as targets of thermally and photoactivated enediynes has been confirmed at the molecular level. Model studies using a labeled substrate confirmed the efficacy of atom transfer from diyl radicals produced from enediynes to form captodatively stabilized carbon centered aminoacyl radicals, which then undergo either fragmentation or dimerization. To exploit this finding, a family of enediynes was developed using an intramolecular coupling strategy. Derivatives were prepared and used to target specific proteins, showing good correlation between affinity and photoinduced protein degrading activity. The findings have potential applications in the design of artificial chemical proteases and add to our understanding of the mechanism of action of the clinically important enediyne antitumor antibiotics.

Chimeric enediynes composed of a photoactivatable warhead coupled either to a porphyrin or spiroalcohol have been prepared. The molecules underwent photoactivation to produce diaryl radicals paving the way for applications in targeted photodynamic therapy.

A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents underwent activation both with enzymatically active prostate specific antigen and alpha-chymotrypsin, releasing free fluorophore via cleavage of a three-component system.

Electron impact ionization of superfluid helium droplets containing several thousand atoms produces a broad distribution of Hen+ ions that peaks at n = 2 and decreases monotonically toward larger n. In larger droplets (say 105 or more atoms), however, the He4+ signal intensity is anomalously large. We have studied the mechanism for the formation of He4+ ions in large helium droplets by varying the duration of the electron impact excitation pulse. Droplets of different average sizes were generated using the expansion of helium at 20 bars and 9-20 K through a pulsed valve nozzle. The resulting ions were analyzed by time-of-flight mass spectroscopy (TOFMS) and quadrupole mass spectroscopy (QMS). The intensity distributions obtained with the TOFMS technique initially showed much smaller He4+ signals than those obtained using QMS. However, we discovered that the intensity anomaly is associated with the duration of the electron bombardment pulse in the TOFMS instrument. Measurements with different electron bombardment pulse durations enabled us to discern a characteristic time of ∼10 μs for enhanced He4+ production in large droplets under our experimental conditions. A qualitative model is presented in which metastables interact on droplet surfaces, yielding two He2+ cores that share a Rydberg electron while minimizing repulsion between the cores. This is the He4+(4A2) state suggested by Knowles and Murrell.

SB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl, leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS.

The effects of hydrogen in Ni(100) submonolayer homoepitaxy have been investigated by classical-potential total-energy calculations with semiclassical hydrogen zero-point-energy corrections. The fast diffusion of H on a flat Ni(100) surface is found to persist even when the surface is decorated with various intrinsic defects such as Ni adatoms, islands, and steps created during epitaxial growth. On the other hand, the presence of H significantly enhances the mobility of both Ni adatoms and Ni islands, resulting in a decrease in the Ni island density and a corresponding increase in the average island size. These results suggest that hydrogen will function as an antisurfactant in Ni(100) homoepitaxy. Connections to available experiments are made. {copyright} {ital 1997} {ital The American Physical Society}

The reduction in pacemaker size coupled with the addition of more current demanding functions has motivated the development of batteries that can supply higher current densities at useful voltages than the lithium/iodine batteries in use today while retaining the volumetric energy density of that system. The lithium/CFx system offers an attractive alternative for advanced pacemaker systems. The battery can deliver currents in the milliampere range without significant voltage drop. The system is compatible with titanium casing, allowing a 50% reduction in weight over the same size lithium/iodine battery. Cells have been designed and tested in these laboratories and have been shown to be suitable for advanced pacemaker applications.

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2 degrees C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1 degree C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.

Silver clusters were assembled in helium droplets of different sizes ranging from 105 to 1010 atoms. The absorption of the clusters was studied upon laser irradiation at 355 nm and 532 nm, which is close to the plasmon resonance maximum in spherical Ag clusters and in the range of the absorption of the complex, branched Ag clusters, respectively. The absorption of the pulsed (7 ns) radiation at 532 nm shows some pronounced saturation effects, absent upon the continuous irradiation. This phenomenon has been discussed in terms of the melting of the complex Ag clusters at high laser fluence, resulting in a loss of the 532 nm absorption. Estimates of the heat transfer also indicate that a bubble may be formed around the hot cluster at high fluences, which may result in ejection of the cluster from the droplet, or disintegration of the droplet entirely.

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies. © 1995, The Biophysical Society. All rights reserved.

Time-resolved Stokes shift measurements (approximately 20 ps effective resolution) are used to measure the solvation times of 16 different probe solutes in a single solvent, 1-propanol (253 K). The solutes were chosen so as to provide a wide range of chemical structures and physical properties consistent with the requirements of strong fluorescence and high solvent sensitivity. Rather than exhibiting a continuous range of solvation times, the dynamics observed with these solutes indicated a division into two distinct classes. The majority (11) of the solutes were observed to fall into a `normal` category. The solvation times of these solutes span a relatively narrow range, 0.14-0.22 ns, times consistent with expectations based on nonspecific theories of solvation dynamics. The remaining five solutes, all simple aromatic amines, form a distinct group whose solvation dynamics is at least 2-fold faster than those of the `normal` solutes. The difference between these two classes of solutes appears to reflect differences in the nature of their hydrogen-bonding interactions with the solvent. 37 refs., 5 figs., 3 tabs.

Accurate measurement of the composition of oil-water emulsions within the process environment is a challenging problem in the oil industry. Ultrasonic techniques are promising because they are non-invasive and can penetrate optically opaque mixtures. This paper presents a method of determining the volume fractions of two immiscible fluids in a homogenized two-phase flow by measuring the speed of sound through the composite fluid along with the instantaneous temperature. Two separate algorithms are developed by representing the composite density as (i) a linear combination of the two densities, and (ii) a non-linear fractional formulation. Both methods lead to a quadratic equation with temperature dependent coefficients, the root of which yields the volume fraction. The densities and sound speeds are calibrated at various temperatures for each fluid component, and the fitted polynomial is used in the final algorithm. We present results when the new algorithm is applied to mixtures of crude oil and process water from two different oil fields, and a comparison of our results with a Coriolis meter; the difference between mean values is less than 1%. Analytical and numerical studies of sensitivity of the calculated volume fraction to temperature changes and calibration errors are also presented.

A previous study showed that several multivalent galactose-specific lectins including the 14-kDa lectin from calf spleen and the lectins from Erythrina indica, Erythrina cristagalli, and soybean agglutinin formed specific cross-linked complexes with the glycoprotein asialofetuin (ASF) [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472]. In the present study, we have used quantitative precipitation analysis to compare the cross-linking activities of the Gal/GalNAc-specific lectin from Erythrina corallodendron (ECorL) and the recombinant protein (rECorL) which lacks the covalently linked heptasaccharide chains of the native lectin, with ASF. At low concentrations of ASF relative to the lectin, native dimeric ECorL binds to each of the three terminal Gal residues of the three N-linked triantennary chains of ASF and precipitates as a cross-linked complex at a ratio of 1:9 ASF/lectin (monomer). With increasing concentrations of ASF, the 1:9 complex changes to a 1:3 ASF/lectin complex, and at higher ASF concentrations, a 1:1 cross-linked complex forms. However, rECorL, which possesses the same specificity and binding affinity as the native lectin, forms only the 1:9 and 1:3 ASF/lectin complexes. Other Erythrina lectins examined, all of which have covalently attached carbohydrate and are structurally similar to ECorL, show the same cross-linking behavior as native ECorL. On the other hand, the dimeric 14-kDa calf spleen lectin which lacks covalently attached carbohydrate forms only 1:9 and 1:3 cross-linked complexes with ASF [Mandal, D. K., & Brewer, C. F. (1992) Biochemistry 31, 8465-8472].(ABSTRACT TRUNCATED AT 250 WORDS)

Since the implantation of the first lithium-powered pacemaker in 1972, biomedical devices powered by lithium batteries have played a significant role in saving lives and providing health-improving therapy. Today a wide variety of devices performing functions from managing cardiac rhythm to relieving pain and administering drugs is available to clinicians. Newer devices such as ventricular assist devices and implantable hearing devices are powered by lithium ion secondary batteries.

The authors have studied the solvent dependence of the steady-state absorption, emission, and time-resolved emission of 7-azaindole (7AI) and two nonreactive analogues, N-methyl-7-azaindole (NMAI) and 7-methyl-7H-pyrrolo[2,3-b]pyridine (7MPP), in an effort to understand the apparently anomalous behavior of 7AI in water. The author`s find that 7AI undergoes the same tautomerization reaction via solvent-catalyzed double proton transfer in water as it does in alcohol solvents. Kinetic modeling shows that the unusual features of the 7AI emission in water arise mainly from quantitative changes in two key rate parameters. In water the rate constant for tautomerization (H{sub 2}O 1.2 x 10{sup 9} s{sup {minus}1} at 24 {degrees}C) is much slower and simultaneously the nonradiative decay rate of the product is much faster (5 x 10{sup 9} s{sup {minus}1} in both H{sub 2}O and D{sub 2}O) than in most alcohols, making observation of the reaction difficult. The reason the reaction rate in water is unusually slow appears to result from differences in the hydrogen bonding structure and dynamics of water compared to monoalcohols. 43 refs., 12 figs., 4 tabs.

The ketone (+/-)-5, which embodies the bicyclic core associated with the title tRNA synthetase inhibitors 1 and 2, has been prepared via a three-component coupling reaction involving 2-(hydroxymethyl)cyclopent-2-enone (15), methylamine (6) and propiolamide (10); straightforward elaboration of the readily derived acetates (-)-21 and (+)-21 has provided the biologically active analogues 23 and 24, respectively, of the title compounds.

The authors have studied the solvent dependence of the steady-state absorption, emission, and time-resolved emission of 7-azaindole (7AI) and two nonreactive analogues, N-methyl-7-azaindole (NMAI) and 7-methyl-7H-pyrrolo[2,3-b]pyridine (7MPP), in an effort to understand the apparently anomalous behavior of 7AI in water. The author`s find that 7AI undergoes the same tautomerization reaction via solvent-catalyzed double proton transfer in water as it does in alcohol solvents. Kinetic modeling shows that the unusual features of the 7AI emission in water arise mainly from quantitative changes in two key rate parameters. In water the rate constant for tautomerization (H{sub 2}O 1.2 x 10{sup 9} s{sup {minus}1} at 24 {degrees}C) is much slower and simultaneously the nonradiative decay rate of the product is much faster (5 x 10{sup 9} s{sup {minus}1} in both H{sub 2}O and D{sub 2}O) than in most alcohols, making observation of the reaction difficult. The reason the reaction rate in water is unusually slow appears to result from differences in the hydrogen bonding structure and dynamics of water compared to monoalcohols. 43 refs., 12 figs., 4 tabs.

Crystalline (monomeric) soybean and (tetrameric) sweet potato beta-amylase were shown to catalyze the cis hydration of maltal (alpha-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form beta-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D2O by soybean beta-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (VH/VD = 6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-[2(a)-2H]maltose as product. These results indicate (for each beta-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. This is a different stereochemistry than reported for starch hydrolysis. With the hydration catalyzed in H2O and analyzed by gas-liquid chromatography, both sweet potato and soybean beta-amylase were found to convert maltal to the beta-anomer of 2-deoxymaltose. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that beta-amylase protonates maltal from a direction opposite that assumed for protonating starch, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures in dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca V ) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca V 2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine ( Sca6 84Q ) developed progressive motor impairment and aggregation of mutant Ca V 2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca 2+ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6 84Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca V 2.1 channels.

Solvation times in three homologous amides, N-methylformamide, N-methylacetamide, and N-methylpropionamide, have been detd. from measurements of the dynamic Stokes shift of the fluorescence spectra of two-probe solutes, prodan and coumarin 102. Single-particle reorientation times have also been measured in one of these solvents, N-methylformamide, by using NMR methods. The solvation dynamics are compared to two theoretical models, the simple continuum model and the dynamic MSA model. Although neither model predicts the time dependence of the response satisfactorily, the average solvation times observed are close to the solvent longitudinal relaxation time ({tau}{sub L}) predicted by the simple continuum model. In contrast, the predictions of the dynamical MSA model are approximately 4 times slower than the observed solvation response.

© 2015 Author(s). Lensless x-ray microscopy requires the recovery of the phase of the radiation scattered from a specimen. Here, we demonstrate a de novo phase retrieval technique by encapsulating an object in a superfluid helium nanodroplet, which provides both a physical support and an approximate scattering phase for the iterative image reconstruction. The technique is robust, fast-converging, and yields the complex density of the immersed object. Images of xenon clusters embedded in superfluid helium droplets reveal transient configurations of quantum vortices in this fragile system.

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.

Nicotinic acetylcholine receptors (nAChRs) are inhibited by several drugs that are commonly thought to be specific for L-type calcium channels (LTCCs). In neurons, LTCCs are activated by nicotine-induced depolarization to engage downstream signaling events; however, the role of LTCC drug interactions with nAChRs in signaling has not been examined in detail. We investigated the effects of LTCC ligands on nAChR currents and downstream signaling in rat superior cervical ganglion (SCG) neurons. We found that 10 μM nicotine and 40 mM K + both reversibly depolarize SCG neurons to −20 mV, sufficient to activate LTCCs and downstream signaling, including induction of nuclear phospho-CREB (pCREB); this induction was blocked by LTCC antagonists. Interestingly, the effects of LTCC antagonists on nicotine-induced signaling to CREB are not mediated by their actions on LTCCs, but rather via inhibition of nAChRs, which prevents nicotine-induced depolarization. We show that this effect is sufficient to block pCREB induction in neurons expressing an antagonist-insensitive LTCC. Taken together, our data show that, at concentrations typically used to block LTCCs, these antagonists inhibit nAChR currents and downstream signaling. These findings serve as a caution in attributing a role for LTCCs when using these drugs experimentally or therapeutically.

A family of differentially substituted poly(ethyleneglycol) building blocks has been assembled from commercially available material. Their utility is demonstrated by formation of amino acid conjugates, image contrast agents, gold nanoparticles, and functional antibody conjugates. Application in the cellular trafficking of antitumoral agent conjugates is expected.

A mild and efficient method for preparation of GlcNAc derivatives by reaction of glycosyl acceptors with glycosyl oxazolines has been developed. The key feature is use of Yb(OTf) 3 as promoter, requiring moderate reaction times and equivalence stoichiometry. We anticipate application in the preparation of a wide range of derivatives of this biologically important class of building blocks.

One of the approaches to fabrication of MEMS involves surface micromachining to define dies on single crystal silicon wafers, dicing of the wafers to separate the dies, and electronic packaging of the individual dies. Dicing and packaging of MEMS accounts for a large fraction of the fabrication costs, therefore, nondestructive evaluation at the wafer level, before dicing, can have significant implications on improving production yield and costs. In this paper, advances in development of optoelectronic holography (OEH) techniques for nondestructive, noninvasive, full-field of view evaluation of MEMS at the wafer level are described. With OEH techniques, quantitative measurements of shape and deformation of MEMS, as related to their performance and integrity, are obtained with sub-micrometer spatial resolution and nanometer measuring accuracy. To inspect an entire wafer with OEH methodologies, measurements of overlapping regions of interest (ROI) on a wafer are recorded and adjacent ROIs are stitched together through efficient 3D correlation analysis algorithms. Capabilities of the OEH techniques are illustrated with representative applications, including determination of optimal inspection conditions to minimize inspection time while achieving sufficient levels of accuracy and resolution.

To increase the targeting potential, circulation time, and the flexibility of surface-attached biomedically-relevant ligands on gold nanoparticles, hetero-bifunctional poly(ethylene glycol) (PEG, MW 1, 500) was synthesized having a thiol group on one terminus and a reactive functional group on the other. Coumarin, a model fluorescent dye, was conjugated to the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreases the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within one hour of incubation.

One strategy for isolating neuronal L-type calcium (Ca(2+)) currents, which typically comprise a minority of the whole cell current in neurons, has been to use pharmacological agents that increase channel activity. This study examines the effects of the benzoyl pyrrole FPL 64176 (FPL) on L-type Ca(2+) currents and compares them to those of the dihydropyridine (+)-202-791. At micromolar concentrations, both agonists increased whole cell current amplitude in PC12 cells. However, FPL also significantly slowed the rate of activation and elicited a longer-lasting slow component of the tail current compared to (+)-202-791. In single channel cell-attached patch recordings, FPL increased open probability, first latency, mean closed time and mean open time more than (+)-202-791, with no difference in unitary conductance. These gating differences suggest that, compared to (+)-202-791, FPL decreases transition rates between open and closed conformations. Where examined, the actions of FPL and (+)-202-791 on whole cell L-type currents in sympathetic neurons appeared similar to those in PC12 cells. In contrast to its effects on L-type current, 10 microM FPL inhibited the majority of the whole cell current in HEK cells expressing a recombinant N-type Ca(2+) channel, raising caution concerning the use of FPL as a selective L-type Ca(2+) channel agonist in neurons.

Twenty two analogues of SB-203207 have been prepared by total synthesis, and evaluated as inhibitors of a range of tRNA synthetases. Changes to the bicyclic core, removing either the terminal amino substituent or the sulfonyl group from the side chain, and altering either the carbon skeleton or stereochemistry of the isoleucine residue, decreases the potency of inhibition of isoleucyl tRNA synthetase. Substituting the isoleucine residue with other amino acids produces inhibitors of the corresponding synthetases. In particular, a methionine derivative is 50-100 times more potent against methionyl tRNA synthetase than against any of the corresponding isoleucyl, leucyl, valyl, alanyl and prolyl synthetases.

We examined the effects of arachidonic acid (AA) on whole cell Ca2+channel activity in rat superior cervical ganglion neurons. Our companion paper (Liu L, Barrett CF, and Rittenhouse AR. Am J Physiol Cell Physiol 280: C1293–C1305, 2001) demonstrates that AA induces several effects, including enhancement of current amplitude at negative voltages, and increased activation kinetics. This study examines the mechanisms underlying these effects. First, enhancement is rapidly reversible by bath application of BSA. Second, enhancement appears to occur extracellularly, since intracellular albumin was without effect on enhancement, and bath-applied arachidonoyl coenzyme A, an amphiphilic AA analog that cannot cross the cell membrane, mimicked enhancement. In addition, enhancement is voltage dependent, in that currents were enhanced to the greatest degree at −10 mV, whereas virtually no enhancement occurred positive of +30 mV. We also demonstrate that AA-induced increases in activation kinetics are correlated with enhancement of current amplitude. An observed increase in the voltage sensitivity may underlie these effects. Finally, the majority of enhancement is mediated through N-type current, thus providing the first demonstration that this current type can be enhanced by AA.

We recently reported that arachidonic acid (AA) inhibits L- and N-type Ca2+currents at positive test potentials in the presence of the dihydropyridine L-type Ca2+channel agonist (+)-202-791 in dissociated neonatal rat superior cervical ganglion neurons [Liu L and Rittenhouse AR. J Physiol (Lond) 525: 291–404, 2000]. In this first of two companion papers, we characterized the mechanism of inhibition by AA at the whole cell level. In the presence of either ω-conotoxin GVIA or nimodipine, AA decreased current amplitude, confirming that L- and N-type currents, respectively, were inhibited. AA-induced inhibition was concentration dependent and reversible with an albumin-containing wash solution, but appears independent of AA metabolism and G protein activity. In characterizing inhibition, an AA-induced enhancement of current amplitude was revealed that occurred primarily at negative test potentials. Cell dialysis with albumin minimized inhibition but had little effect on enhancement, suggesting that AA has distinct sites of action. We examined AA's actions on current kinetics and found that AA increased holding potential-dependent inactivation. AA also enhanced the rate of N-type current activation. These findings indicate that AA causes multiple changes in sympathetic Ca2+currents.

The lithium/iodine-polyvinylpyridine battery, first implanted 20 years ago, has become the power source of choice for the cardiac pacemaker. Over the last 20 years, improvements in cell chemistry, cell design, and modeling of cell performance have been made. Cells today exhibit an energy density over three times as great as cells produced in 1972. Well over 2 million pacemakers have been implanted with this chemistry, and the system has exhibited excellent reliability.

Highly focused pulsed laser microbeams can be used to precisely dissect, inactivate, or perturb cells and subcellular targets. Here we introduce a new technique which employs pulsed microbeams to transiently permeabilize the plasma cell membrane and affect the delivery of molecules from the extracellular environment into the cell. This optically produced cell permeabilization can be applied using non- specific or specific modalities. In the non-specific modality, which we term 'optoporation,' the pulsed microbeam is focused onto the glass coverslip on which the cells are plated. The generation of mechanical transients in connection with irradiation of the glass achieves molecular delivery to a number of cells proximal to the irradiation site. In the specific modality, termed 'optoinjection,' the microbeam is focused directly onto the plasma cell membrane and achieves molecular delivery into that cell alone. To quantify the irradiation geometry involved in these and other microbeam processes, as well as examine the possibility of certain non- linear effects, we have developed a system using photochromic polymer films to characterize microbeam propagation and its effects within microirradiated targets. These photochromic polymers confirm that the laser microbeam are indeed focused to submicron dimensions within the targets in our systems. In addition the behavior of such polymers at higher pulse energies and irradiances indicate that multiphoton absorption and/or plasma formation may mediate some laser microirradiation processes.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The purpose of this study was to determine whether in vivo fluorescence detection of protoporphyrin IX (PpIX) could be used to identify intraperitoneal micrometastases of epithelial ovarian carcinoma after application of 5-aminolevulinic acid (ALA). ALA was applied intraperitoneal at different concentrations (25, 50, and 100 mg/kg) and iv (100 mg/kg) to immunocompetent Fischer 344 rats bearing a syngeneic epithelial ovarian carcinoma. At different time intervals after ALA administration (1.5, 3, and 6 hr) the peritoneal cavity was illuminated with ultraviolet (uv) light. In vivo fluorescence of PpIX initially was determined by direct visualization. Subsequently ex vivo measurements were made with a slow-scan, thermoelectrically cooled CCD camera. Red in vivo fluorescence was observed in ovarian micrometastases smaller than 0.5 mm in 100% of the ALA-administered animals independent of time interval, drug concentration, or route of administration. The intensity of the fluorescence was concentration dependent as strong fluorescence was consistently found only above 25 mg/kg ALA. Ex vivo tumor to peritoneum fluorescence yield peaked 3 hr after administration of a 100 mg/kg intraperitoneal dose. Direct visualization of in vivo fluorescence after ALA application may improve the detection of intraperitoneal ovarian cancer micrometastases.

The stereochemistry of the indium-promoted reaction of allyl bromide with alpha-thia (PhS and MeS), disubstituted alpha-amino (Bn(2)N, Me(2)N, isoindolyl), and protected alpha-amino aldehydes (Ac and Boc) in water has been evaluated. The reactions involving the sulfur derivatives are minimally diastereoselective, indicating that the allylindium reagent is not thiophilic. Chelation is not observed and pi-facial discrimination is achieved via Felkin-Ahn transition states under the steric control of the substituents. The Garner aldehyde is also anti-diastereoselective. Interestingly, N-acetylmannosamine is appreciably responsive to chelation control and is capable of generating 90% of the syn beta-amino alcohol when reacted in a 0.5 M NH(4)Cl solution. While the alpha-dibenzylamino substituent is too bulky to enter into complexation, the alpha-dimethylamino group is not and can lead to high levels (99%) of syn diastereomer. The size of other neighboring substituents does have an impact on pi-facial discrimination in these systems and can erode the stereoselectivity accordingly.

The ketone (+/-)-5, which embodies the bicyclic core associated with the title tRNA synthetase inhibitors 1 and 2, has been prepared via a three-component coupling reaction involving 2-(hydroxymethyl)cyclopent-2-enone (15), methylamine (6) and propiolamide (10); straightforward elaboration of the readily derived acetates (-)-21 and (+)-21 has provided the biologically active analogues 23 and 24, respectively, of the title compounds.