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Selected AbstractsAn in vivo comparison of photoactivatable fluorescent proteins in an avian embryo modelDEVELOPMENTAL DYNAMICS, Issue 6 2007Danny A. Stark Abstract Tracing the lineage or neighbor relationships of cells in a migratory population or deep within an embryo is difficult with current methods. The recent explosion of photoactivatable fluorescent proteins (PAFPs) offers a unique cell labeling tool kit, yet their in vivo performance in intact embryos and applicability have not been thoroughly explored. We report a comparison study of PAGFP, PSCFP2, KikGR, and Kaede analyzed in the avian embryo using confocal and 2-photon microscopy. PAFPs were introduced into the chick neural tube by electroporation and each photoconverted in the neural crest or cells in the neural tube with exposure to 405 nm light, but showed dramatic differences in photoefficiency and photostability when compared at the same 2% laser power. KikGR and Kaede photoconverted with ratios only slightly lower than in vitro results, but cells rapidly photobleached after reaching maximal photoefficiency. PSCFP2 had the lowest photoefficiency and photoconverted nearly 70 times slower than the other dual-color PAFPs tested, but was effective at single-cell marking, especially with 2-photon excitation at 760 nm. The dual-color PAFPs were more effective to monitor cell migratory behaviors, since non-photoconverted neighboring cells were fluorescently marked with a separate color. However, photoconverted cells were limited in all cases to be visually distinguishable for long periods, with PSCFP2 visible from background the longest (48 hr). Thus, photoactivation in embryos has the potential to selectively mark less accessible cells with laser accuracy and may provide an effective means to study cell,cell interactions and short-term cell lineage in developmental and stem cell biology. Developmental Dynamics 236:1583,1594, 2007. © 2007 Wiley-Liss, Inc. [source] Effects of Aging on Interfacial Microstructure and Reliability Between SnAgCu Solder and FeNi/Cu UBM,ADVANCED ENGINEERING MATERIALS, Issue 6 2010Qing-Sheng Zhu Effects of thermal aging on the interfacial microstructure and reliability of the SnAgCu/FeNi-Cu joint are investigated. It is found that aging effects depends strongly on the temperature. Aging at low temperature, e.g., at 125,°C, a submicron meter thick FeSn2 IMC layer formed at the SnAgCu/FeNi-Cu interface during reflowing grows at a rate twenty times slower than the growth rate of the IMC at the SnAgCu/Cu interface. At high temperature, e.g., at 180,°C, the Cu element is found to diffuse through FeNi layer to produce the (Cu, Ni)6Sn5 IMC and this IMC layer grows even faster than the IMC at the SnAgCu/Cu interface. Solder ball shear test results show that the SnAgCu/FeNi-Cu joint has a comparable strength to the SnAgCu/Cu joint after reflowing, and the strength drop after aging at 125,°C is less than that of the SnAgCu/Cu joint. However, after aging at 180,°C, the strength of the SnAgCu/FeNi-Cu joint is degraded to a low value, along with a shift in failure mode from the solder fracture to the brittle intermetallics fracture. [source] Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000Vladimir S. Vorobjev Abstract The modulation of ,-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration,response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was ,,90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 m m glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds. [source] A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgarisFEBS JOURNAL, Issue 1 2002Brian Ó Nuallain The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s,1 in 3 m urea in 25 mm sodium phosphate at 25 °C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein. [source] Timing of Depolarization and Contraction in the Paced Canine Left Ventricle:JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 2003Experiment, Model Introduction: For efficient pump function, contraction of the heart should be as synchronous as possible. Ventricular pacing induces asynchrony of depolarization and contraction. The degree of asynchrony depends on the position of the pacing electrode. The aim of this study was to extend an existing numerical model of electromechanics in the left ventricle (LV) to the application of ventricular pacing. With the model, the relation between pacing site and patterns of depolarization and contraction was investigated. Methods and Results: The LV was approximated by a thick-walled ellipsoid with a realistic myofiber orientation. Propagation of the depolarization wave was described by the eikonal-diffusion equation, in which five parameters play a role: myocardial and subendocardial velocity of wave propagation along the myofiber cm and ce; myocardial and subendocardial anisotropy am and ae; and parameter k, describing the influence of wave curvature on wave velocity. Parameters cm, ae, and k were taken from literature. Parameters am and ce were estimated by fitting the model to experimental data, obtained by pacing the canine left ventricular free wall (LVFW). The best fit was found with cm= 0.75 m/s, ce= 1.3 m/s, am= 2.5, ae= 1.5, and k= 2.1 × 10,4 m2/s. With these parameter settings, for right ventricular apex (RVA) pacing, the depolarization times were realistically simulated as also shown by the wavefronts and the time needed to activate the LVFW. The moment of depolarization was used to initiate myofiber contraction in a model of LV mechanics. For both pacing situations, mid-wall circumferential strains and onset of myofiber shortening were obtained. Conclusion: With a relatively simple model setup, simulated depolarization timing patterns agreed with measurements for pacing at the LVFW and RVA in an LV. Myocardial cross-fiber wave velocity is estimated to be 0.40 times the velocity along the myofiber direction (0.75 m/s). Subendocardial wave velocity is about 1.7 times faster than in the rest of the myocardium, but about 3 times slower than as found in Purkinje fibers. Furthermore, model and experiment agreed in the following respects. (1) Ventricular pacing decreased both systolic pressure and ejection fraction relative to natural sinus rhythm. (2) In early depolarized regions, early shortening was observed in the isovolumic contraction phase; in late depolarized regions, myofibers were stretched in this phase. Maps showing timing of onset of shortening were similar to previously measured maps in which wave velocity of contraction appeared similar to that of depolarization. (J Cardiovasc Electrophysiol, Vol. 14, pp. S188-S195, October 2003, Suppl.) [source] The Lifetimes of Pharaonis Phoborhodopsin Signaling States Depend on the Rates of Proton Transfers,Effects of Hydrostatic Pressure and Stopped Flow Experiments,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Takashi Kikukawa Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate. [source] Long-distance transport of gases in plants: a perspective on internal aeration and radial oxygen loss from rootsPLANT CELL & ENVIRONMENT, Issue 1 2003T. D. COLMER ABSTRACT Internal transport of gases is crucial for vascular plants inhabiting aquatic, wetland or flood-prone environments. Diffusivity of gases in water is approximately 10 000 times slower than in air; thus direct exchange of gases between submerged tissues and the environment is strongly impeded. Aerenchyma provides a low-resistance internal pathway for gas transport between shoot and root extremities. By this pathway, O2 is supplied to the roots and rhizosphere, while CO2, ethylene, and methane move from the soil to the shoots and atmosphere. Diffusion is the mechanism by which gases move within roots of all plant species, but significant pressurized through-flow occurs in stems and rhizomes of several emergent and floating-leaved wetland plants. Through-flows can raise O2 concentrations in the rhizomes close to ambient levels. In general, rates of flow are determined by plant characteristics such as capacity to generate positive pressures in shoot tissues, and resistance to flow in the aerenchyma, as well as environmental conditions affecting leaf-to-air gradients in humidity and temperature. O2 diffusion in roots is influenced by anatomical, morphological and physiological characteristics, and environmental conditions. Roots of many (but not all) wetland species contain large volumes of aerenchyma (e.g. root porosity can reach 55%), while a barrier impermeable to radial O2 loss (ROL) often occurs in basal zones. These traits act synergistically to enhance the amount of O2 diffusing to the root apex and enable the development of an aerobic rhizosphere around the root tip, which enhances root penetration into anaerobic substrates. The barrier to ROL in roots of some species is induced by growth in stagnant conditions, whereas it is constitutive in others. An inducible change in the resistance to O2 across the hypodermis/exodermis is hypothesized to be of adaptive significance to plants inhabiting transiently waterlogged soils. Knowledge on the anatomical basis of the barrier to ROL in various species is scant. Nevertheless, it has been suggested that the barrier may also impede influx of: (i) soil-derived gases, such as CO2, methane, and ethylene; (ii) potentially toxic substances (e.g. reduced metal ions) often present in waterlogged soils; and (iii) nutrients and water. Lateral roots, that remain permeable to O2, may be the main surface for exchange of substances between the roots and rhizosphere in wetland species. Further work is required to determine whether diversity in structure and function in roots of wetland species can be related to various niche habitats. [source] Experimental determinations of factors affecting the sink rates of baited hooks to minimize seabird mortality in pelagic longline fisheriesAQUATIC CONSERVATION: MARINE AND FRESHWATER ECOSYSTEMS, Issue 6 2010Graham Robertson Abstract 1.An experiment was conducted in Australia's pelagic longline fishery to establish a scientific basis for the introduction of line weighting to reduce seabird mortality. The experiment examined the effects of different bait species (blue mackerel, yellow-tail mackerel and squid), bait life status (dead or alive), weight of leaded swivels (60,g, 100,g and 160,g) and leader length (distance between leaded swivel and hooks: 2,m, 3,m and 4,m) on the sink rates of baited hooks from 0,6,m deep. 2.On average, live bait sank much more slowly than dead bait. The sink rates of individual live bait were highly variable: many were <2,m underwater 18,s after deployment, including some on the heaviest swivels, and some were <10,m deep after 120,s. 3.Within the dead bait group, all three swivel weights on 3,m and 4,m leaders sank at similar rates. Initial sink rates (e.g. 0,2,m) were 2,3 times slower than final rates (e.g. 4,6,m) for all combinations of swivel weight and leader length. The fastest initial and final sink rates were associated with heavy swivels placed close to hooks. 4.The results show that (a) compared with dead bait, live bait greatly increases the exposure of baited hooks to seabirds; (b) initial sink rates of dead bait are increased by placing leaded swivels close to hooks and final rates by increasing the weight of the swivels; (c) adding weight to long leaders makes little difference to sink rates; and (d) the small (incremental) changes to swivel weights and leader lengths typically preferred by industry will be difficult to detect at sea and unlikely to substantially reduce seabird mortality. 5.We suggest that experiments designed to reduce seabird mortality from that associated with 60,g swivels and ,3.5,m leaders (the preferred option by industry) should aim to expedite the initial sink rates as well as rates to deeper depths. This objective could be achieved by including branch lines with ,120,g swivels ,2,m in comparative assessments of the effectiveness of line weighting regimes in reducing seabird mortality. Copyright © 2010 John Wiley & Sons, Ltd. [source] Structure and Dynamics of Water Confined in Dimethyl SulfoxideCHEMPHYSCHEM, Issue 1 2006A. Wulf Abstract We study the structure and dynamics of hydrogen-bonded complexes of H2O/D2O and dimethyl sulfoxide (DMSO) by infrared spectroscopy, NMR spectroscopy and ab initio calculations. We find that single water molecules occur in two configurations. For one half of the water monomers both OH/OD groups form strong hydrogen bonds to DMSO molecules, whereas for the other half only one of the two OH/OD groups is hydrogen-bonded to a solvent molecule. The H-bond strength between water and DMSO is in the order of that in bulk water. NMR deuteron relaxation rates and calculated deuteron quadrupole coupling constants yield rotational correlation times of water. The molecular reorientation of water monomers in DMSO is two-and-a-half times slower than in bulk water. This result can be explained by local structure behavior. [source] | |||||||||||||