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Electrophysiologic studies have characterized ion channels in lacrimal gland acinar cells, but due to relative paucity and inaccessibility, such studies on lacrimal gland duct cells are challenging. The duct cells are believed to secrete the high level of K+ that is present in tears. The goal of this project was to develop a method for isolation of viable, single duct cells, demonstrate their utility for patch-clamp recording and characterize the K+ channels expressed by duct cells. Exorbital lacrimal gland slices from Sprague-Dawley rats were incubated with collagenase. Under a microscope, the ducts were microdissected from the glands and then incubated with elastase and collagenase. Dispersed duct cells were plated on a cover slip coated with BD Cell-Tak. Duct cells were distinguished from acinar cells by their smaller size and lack of granularity. Whole-cell K+ currents were recorded from duct cells using the perforated-patch technique and pipettes with resistances of less than 10 MΩ. EGTA and CaCl2 in the pipette solution were adjusted to give 1 uM free Ca++. When held at −80 mV, duct cells showed K+ currents that activated at command voltages near 0 mV and reached amplitudes near 1 nA at +100 mV. Currents reached peak amplitude less than 20 ms after depolarization and did not inactivate. These currents were inactivated by holding the cells at 0 mV. Currents were blocked reversibly by TEA in the presence of Ca2+, but were not blocked by TEA in the absence of Ca2+. Currents were not affected by clotrimazole (10 uM) or Ba2+ (5 mM). A method has been established for isolation and dissociation lacrimal gland duct cells for electrophysiologic studies. These cells express a voltage-activated K+ channel that is dependent on the presence of intracellular Ca2+ and may correspond to the IKCa1 channel expressed on the apical membranes of lacrimal gland ducts.

The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K+ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm2 UVB. K+ channel activation and loss of intracellular K+ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K+ channel activation and loss of intracellular K+ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K+ channel activation and decreased loss of intracellular K+ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K+ channel activation and loss of intracellular K+. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm2 UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the observation that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits the UVB-induced K+ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K+ channel activation.

UVB exposure at ambient outdoor levels triggers rapid K(+) loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K(+), but considerably less apoptosis occurs when the medium contains the high K(+) concentration that is present in tears (25 mM). Since Ba(2+) blocks several K(+) channels, we tested whether Ba(2+)-sensitive K(+) channels are responsible for some or all of the UVB-activated K(+) loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm(2). Patch-clamp recording was used to measure UVB-induced K(+) currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba(2+). K(+) currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba(2+). When HCLE cells were incubated with 5 mM Ba(2+) after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K(+) current activation and loss of intracellular K(+) leads to activation of the caspase cascade and apoptosis. Extracellular Ba(2+) inhibits UVB-induced apoptosis by preventing loss of intracellular K(+) when K(+) channels are activated. Ba(2+) therefore has effects similar to elevated extracellular K(+) in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K(+) in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB.

Industrialized society will transition away from dependence on non-renewable resources (fossil fuels, in particular) sometime in the foreseeable future. How disruptive this transition will be to the economy and societal well-being is unknown, particularly if there are any sudden resource supply constraints. However, the effects of resource supply constraints on an economy, or models of the interdependent relationship between the economy and natural capital overall have not been thoroughly developed. One problem is that traditional models of the economy assume linear growth, while highly interdependent industrialized economies may behave more like a complex adaptive system with non-linear, path-dependent, and unexpected growth trajectories. Agent-based models have been shown to successfully model important aspects of a complex adaptive economy. This paper uses an agent-based model to demonstrate potential economic impacts for industrialized economies in the face of a sudden resource supply constraint. Economic “agents” mine resources and invent technology. Through trade and specialization, the economy evolves from a collection of self-sustaining, resource-poor agents to a society with a high degree of interdependence and wealth. Economic growth, however, comes with a cost; the interdependence that arises from specialization and trade also leads to a less resilient economy. Unexpected, large economic collapse can arise from a shock to even a single resource, due to each resource’s interdependent role in the economy.

The goal of this study was to investigate the efflux of K + from human corneal limbal epithelial cells (HCLE) exposed to ambient levels of UVB, which is known to cause apoptosis, and to examine the effect of K + channel blockers on loss of potassium induced by UVB. HCLE cells were exposed to 100–200 mJ/cm 2 UVB, followed by incubation in culture media with 5.5–100 mM K + , BDS-1, Ba 2+ or ouabain. To measure intracellular cations, cells were washed in 280 mM sucrose and lysed in DI water. K + and Na + levels in lysates were measured by ion chromatography. HCLE cells showed maximal loss of K + i 10 min after exposure to UVB and 5.5 mM K + media, with recovery of normal K + levels after 90 min. Treatment with 1 μM BDS-1 following UVB exposure reduced the loss of K + by HCLE cells. Exposure to 0.1–5 mM Ba 2+ inhibited UVB-induced K + loss in a time and dose-dependent manner. These results confirm that blocking K + channels in HCLE cells exposed to UVB prevents efflux of K + , confirming that UVB activates K + channels in these cells. Electrophysiology data show that K + channels remain highly active at least 90 min after UVB exposure. HCLE cells exposed to UVB and incubated in 0.01–1 μM ouabain did not recover from UVB-induced K + loss. These data suggest that the Na/K pump may act to restore [K + ] i to control levels in HCLE cells following UVB exposure and that the pump is not damaged by exposure to UVB. Incubation of HCLE cells exposed to UVB in medium with 25–100 mM K + media prevented K + efflux at extracellular concentrations as low as 25 mM (the concentration in tear fluid), maintaining control levels of K + i . In all experiments inward fluxes and intracellular Na + levels mirrored K + changes, albeit at the expected lower concentrations. The prevention of UVB-induced K i + loss by 25 mM K o + is consistent with the possible contribution of the relatively high K + concentration in tears to protection of the corneal epithelium from ambient UVB.

The goal of this study was to determine if the high [K + ] in tears, 20–25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal–limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50–200 mJ/cm 2 followed by measurement of K + channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K + channels after UV-B exposure at 80 mJ/cm 2 or 150 mJ/cm 2 and a decrease in UV-induced K + efflux with increasing [K + ] o . The UV-activated current was partially blocked by the specific K + channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100–200 mJ/cm 2 . DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100 mM K o + after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50–200 mJ/cm 2 , but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100 mM K o + following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100–200 mJ/cm 2 . This decrease in mitochondrial potential was prevented by 100 mM K o + ; however, 25 or 50 mM K o + provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50–200 mJ/cm 2 , and this UV-activation was significantly reduced by 25–100 mM K o + . The data show that the physiologically relevant [K + ] o of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K + ] in tears reduces loss of K + from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular surface from ambient UV radiation.

The goal of this study was to elucidate the pathway by which UVB initiates efflux of K+ and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80–150 mJ/cm2 UVB and incubated in culture medium with 5.5 mM K+. Knockdown of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K+ currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K+ channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf-1 knockdown support the conclusion that the intrinsic pathway is more important in UVB-induced apoptosis in HCLE cells.

We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2–5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve.

A retinal ganglion cell commonly expresses two spatially overlapping receptive field mechanisms. One is the familiar “center/surround,” which sums excitation and inhibition across a region somewhat broader than the ganglion cell's dendritic field. This mechanism responds to a drifting grating by modulating firing at the drift frequency (linear response). Less familiar is the “nonlinear” mechanism, which sums the rectified output of many small subunits that extend for millimeters beyond the dendritic field. This mechanism responds to a contrast-reversing grating by modulating firing at twice the reversal frequency (nonlinear response). We investigated this nonlinear mechanism by presenting visual stimuli to the intact guinea pig retinain vitrowhile recording intracellularly from large brisk and sluggish ganglion cells. A contrast-reversing grating modulated the membrane potential (in addition to the firing rate) at twice the reversal frequency. This response was initially hyperpolarizing for some cells (either ON or OFF center) and initially depolarizing for others. Experiments in which responses to bars were summed in-phase or out-of-phase suggested that the single class of bipolar cells (either ON or OFF) that drives the center/surround response also drives the nonlinear response. Consistent with this, nonlinear responses persisted in OFF ganglion cells when ON bipolar cell responses were blocked byl-AP-4. Nonlinear responses evoked from millimeters beyond the ganglion cell were eliminated by tetrodotoxin. Thus, to relay the response from distant regions of the receptive field requires a spiking interneuron. Nonlinear responses from different regions of the receptive field added linearly.

Many cold positrons in ultrahigh vacuum are required to produce cold antihydrogen, to cool highly stripped ions, and to study ultracold plasmas. Up to 3.5 × 104 such positrons have now been accumulated into the ultrahigh vacuum of a 4.2 K Penning trap, at a rate exceeding 103/hr. Both the accumulation rate (per high energy positron incident at the trap), and the number accumulated, are much larger than ever before realized at low temperatures in high vacuum. Cooling of high energy positrons (from 22Na decay) in a tungsten crystal near the trap, together with purely electronic trapping and damping, are key to the efficient accumulation and to projected improvements.

Positrons from a 12 mCi22Na source are slowed by a W(110) reflection moderator and then captured in a Penning trap, by damping their motion with a tuned circuit. Because of the stability of the Penning trap and the cryogenic ultra-high vacuum environment, we anticipate that positrons can be accumulated and stored indefinitely. A continuous loading rate of 0.14 e+/s is observed for 32 h in this initial demonstration. More than 1.6×104 positrons have thus been trapped and stored at 4 K, with improvements expected. The extremely high vacuum is required for compatibility with an existing antiproton trap, which has already held more than 105 antiprotons at 4 K, for producing antihydrogen at low temperatures. The extremely cold positrons in high vacuum may also prove to be useful for cooling highly stripped ions.

The storage of extremely cold (4 K) antiprotons in a Penning trap is an important step toward the creation and study of cold antihydrogen. The other required ingredient, the largest possible number of comparably cold positrons, is still lacking. These would be recombined in a high vacuum with the trapped antiprotons, already stored at a pressure below 5×10−17 Torr, thereby avoiding annihilation of the antihydrogen atoms before they can be used in high accuracy measurements or in controlled collision experiments. In an exploratory experiment, positrons from a 18 mCi22Na source follow fringing field lines of a 6 T superconducting solenoid through tiny apertures in the electrodes of a Penning trap to strike a tungsten (reflection) moderator. The positron beam is chopped mechanically and a lock-in directly detects a positron current of 2.5×106e+/s on the moderator. The use of a moderator, unlike an earlier experiment in which < 100 positrons were confined in vacuum, should greatly increase the number of positrons trapped in high vacuum.

The possibility to produce, trap and study antihydrogen atoms rests upon the recent availability of extremely cold antiprotons in a Penning trap. Over the last five years, our TRAP Collaboration has slowed, cooled and stored antiprotons at energies 1010 lower than was previously possible. The storage time exceeds 3.4 months despite the extremely low energy, which corresponds to 4.2 K in temperature units. The first example of measurements which become possible with extremely cold antiprotons is a comparison of the antiproton inertial masses which shows they are the same to a fractional accuracy of 4×10−8. (This is 1000 times more accurate than previous comparisons and large additional increases in accuracy are anticipated.) To increase the number of trapped antiprotons available for antihydrogen production, we have demonstrated that we can accumulate or “stack” antiprotons cooled from successive pulsed injections into our trap.

During the last several years, our TRAP collaboration has pioneered techniques for slowing, trapping, cooling and indefinitely storing antiprotons to energies more than 1010 times lower than previously possible. The radio signal from a single trapped antiproton is now being used for precision measurements. Many cold antiprotons are “stacked” as another important step toward the eventual production of antihydrogen, and positrons have been trapped in vacuum.

The antiproton and proton inertial masses have been measured to be the same to a fractional accuracy of 4 × 10−8 by our TRAP collaboration at CERN. Two key experimental advances were employed which have a wider applicability for mass spectroscopy. A high-precision cylindrical trap with open-endcap electrodes allows free access to the trap center for particle loading, and a self-shielding superconducting solenoids system cancels fluctuations in the ambient magnetic fields.

The goal of this study was to determine whether prevention of K(+) loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150-200 mJ/cm(2) UV-B demonstrated induction of apoptosis 6 h after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K(+). If this protection is due to a reduction of UV-induced K(+) loss then K(+) channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm(2) was significantly reduced when the cells were incubated in 0.3 microM BDS-I or 0.05-1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1-0.3 microM BDS-I and 0.1-1 mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 microM BDS-I and 0.01-0.05 mM quinidine. Patch-clamp recording showed activation of K(+) channels after exposure to UV-B and a decrease in outward K(+) current was observed following application of BDS-I. Quinidine did not block K(+) currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K(+) channels. The effect of the K(+) channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K(+) efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K(+)] in tears may protect the corneal epithelium from effects of ambient UV-B.

The receptive field of the Y-ganglion cell comprises two excitatory mechanisms: one integrates linearly over a narrow field, and the other integrates nonlinearly over a wide field. The linear mechanism has been attributed to input from bipolar cells, and the nonlinear mechanism has been attributed to input from a class of amacrine cells whose nonlinear "subunits" extend across the linear receptive field and beyond. However, the central component of the nonlinear mechanism could in theory be driven by bipolar input if that input were rectified. Recording intracellularly from the Y-cell in guinea pig retina, we blocked the peripheral component of the nonlinear mechanism with tetrodotoxin and found the remaining nonlinear receptive field to be precisely co-spatial with the central component of the linear receptive field. Both linear and nonlinear mechanisms were caused by an excitatory postsynaptic potential that reversed near 0 mV. The nonlinear mechanism depended neither on acetylcholine nor on feedback involving GABA or glycine. Thus the central components of the ganglion cell's linear and nonlinear mechanisms are apparently driven by synapses from the same rectifying bipolar cell.

Over the last five years, a series of experiments by our TRAP Collaboration has made it possible to slow, cool and store antiprotons at energies 1010 lower than was previously possible. Despite the extremely low energy, the storage time exceeds 3.4 months. The first example of measurements which become possible with extremely cold antiprotons is a comparison of the antiproton and proton inertial masses which shows they are the same to a fractional accuracy of 4×10−8. This is 1000 times more accurate than previous comparisons and large additional increases in accuracy are anticipated soon. The availability of low energy antiprotons opens other experimental possibilities as well, such as the production and study of trapped antihydrogen atoms. To increase the number of trapped antiprotons we have demonstrated that we can accumulate antiprotons from successive pulsed injections of antiprotons into the trap.

The authors have measured the electrical resistivity, rho , and the thermopower, S, for the liquid alloy MgZn across the entire concentration range. New data for rho and S for pure liquid Zn are also reported. Data for the liquid alloy are shown to be consistent with the Faber-Ziman theory, provided the pseudopotential for Zn is assumed to be energy dependent. A comparison of the data for the liquid alloy with data previously reported for the solid amorphous alloy indicate that this energy dependence should also be considered in the solid.

Since antiprotons have been captured in an ion trap, we consider the possibility of producing antihydrogen by merging cold trapped plasmas of antiprotons and positrons. The calculated, instantaneous rate for antihydrogen production by the 3-body recombination $$p^ - + e^ + + e^ + \to \bar H + e^ + $$ is much higher than for other proposed techniques, opening up intriguing experimental possibilities.

Antiprotons from the Low Energy Antiproton Ring of CERN are slowed from 21 MeV to below 3 keV by being passed through 3 mm of material, mostly Be. While still in flight, the kilo-electron volt antiprotons are captured in a Penning trap created by the sudden application of a 3-kV potential. Antiprotons are held for 100 s and more. Prospects are now excellent for much longer trapping times under better vacuum conditions. This demonstrates the feasibility of a greatly improved measurement of the inertial mass of the antiproton and opens the way to other intriguing experiments. The possibility of producing antihydrogen by merging cold, trapped plasmas of positrons and antiprotons is discussed.

We develop a method of managing strategic reserves, in this case, the U.S. Strategic Cobalt Reserve. A rationale for the stockpiling of cobalt is presented, followed by a method for bringing the stockpiled amount from any level to a desired goal, in this case an amount determined by the Federal Emergency Management Agency to last through three years of conventional warfare. The method involves solving a stochastic programming problem in order to balance the expected values of the social benefit and the social cost of building the stockpile—Social benefit is accrued by decreasing the impact and the probability of a war or a major supply disruption occuring before the stockpile goal is realized; social cost is determined from the additional amount U.S. cobalt consumers must pay due to the increase in world demand brought about by stockpiling. The management of the filled stockpile is then discussed, introducing the idea of using the stockpile to assure stability in the world price of cobalt and of defraying maintenance costs by market speculation. Least-squares fitting is used to determine whether prices are high or low and how much to sell or buy, respectively, to bring prices back into line. Then the conditions under which the stockpile should be drawn down are considered, with the proper rate and total amount of released stockpile material determined for two cases, that of a major supply disruption and that of actual warfare. Finally, generalization of the method to cover other strategic stockpiles is discussed.

Cylindrical Penning traps with open-endcap electrodes are compared with the hyperbolic traps which are currently used for high precision experiments. Cylindrical traps are easier to construct and allow free access to the center of the trap for particle loading, laser beams, or microwaves. The trapping potential can be tuned while particles are inside the trap to improve harmonicity. Special geometries are noted which allow anharmonicities to be tuned out without affecting the trap well depth, and the effects of trap imperfections and gaps between the electrodes are discussed. Such traps will be used for measuring the antiproton mass, for colling of trapped antiprotons, and possibly for producing antihydrogen.

Now that antiprotons have been captured in an ion trap, we investigate the possibility of producing antihydrogen by merging cold trapped plasmas of antiprotons and positrons. The calculated rate for antihydrogen production by the three-body recombination p − + e + + e + → H + e + is many orders of magnitude higher than for any other proposed technique, opening up intriguing experimental possibilities.