Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.     

Particle swarm optimization

Particle swarm optimization (PSO) has undergone many changes since its introduction in 1995. As researchers have learned about the technique, they have derived new versions, developed new applications, and published theoretical studies of the effects of the various parameters and aspects of the algorithm. This paper comprises a snapshot of particle swarming from the authors’ perspective, including variations in the algorithm, current and ongoing research, applications and open problems.     

Adaptation of the dichlorofluorescein assay for detection of radiation-induced oxidative stress in cultured cells

The oxidation of 2'7'-dichlorofluorescin (DCFH) to 2'7'-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to gamma rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with gamma rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.     

Decreased absolute amygdala volume in cocaine addicts

The amygdala is instrumental to a set of brain processes that lead to cocaine consumption, including those that mediate reward and drug craving. This study examined the volumes of the amygdala and hippocampus in cocaine-addicted subjects and matched healthy controls and determined that the amygdala but not the hippocampus was significantly reduced in volume. The right-left amygdala asymmetry in control subjects was absent in the cocaine addicts. Topological analysis of amygdala isosurfaces (population averages) revealed that the isosurface of the cocaine-dependent group undercut the anterior and superior surfaces of the control group, implicating a difference in the corticomedial and basolateral nuclei. In cocaine addicts, amygdala volume did not correlate with any measure of cocaine use. The amygdala symmetry coefficient did correlate with baseline but not cocaine-primed craving. These findings argue for a condition that predisposes the individual to cocaine dependence by affecting the amygdala, or a primary event early in the course of cocaine use.     

Geomorphology and Late Quaternary development of Middleton and Elizabeth Reefs

Middleton and Elizabeth Reefs are two mid-latitude, annular reefs within the Lord Howe linear chain of volcanic islands and seamounts in the southwestern Pacific Ocean. Drilling, vibrocoring, seismic profiling, and dating indicate that each has a rim of Holocene reef framework, enclosing a lagoon partly filled by prograding sand sheets composed of fragments of coral, coralline algae, foraminifers, and other skeletal debris. The reefs lie close to the latitudinal limits for coral growth and the reef framework is very porous, dominated by branching rather than massive corals. Coralline algae are the principal binding agent in the upper reef framework. Holocene reef growth began on a foundation of Pleistocene reefal limestone encountered at a depth of 8 m in cores on the windward side of Middleton Reef. Holocene corals became established on this foundation around 6,700 radiocarbon yr B.P., implying little if any lag after inundation of the platform by the post-glacial sea-level rise. Windward reef growth tracked sea-level rise (keep-up mode), and a prominent reef crest was established on both reefs by 5,000 yr B.P. Leeward margins appear to have been characterized by catch-up growth. Development of cays is limited, and has been restricted by the paucity of coarse coralline debris or cemented conglomerate on which islands could become established. The morphology and development of Middleton and Elizabeth Reefs has been similar to that of tropical atolls, although the rate of subsidence appears to have been relatively slow reflecting their position on the margin of the foundered continental crust of the Lord Howe Rise.     

The structural basis for the selectivity of benzotriazole inhibitors of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) has been implicated in the regulation of the insulin signaling pathway and represents an attractive target for the design of inhibitors in the treatment of type 2 diabetes and obesity. Inspection of the structure of PTP1B indicates that potent PTP1B inhibitors may be obtained by targeting a secondary aryl phosphate-binding site as well as the catalytic site. We report here the crystal structures of PTP1B in complex with first and second generation aryldifluoromethyl-phosphonic acid inhibitors. While all compounds bind in a previously unexploited binding pocket near the primary binding site, the second generation compounds also reach into the secondary binding site, and exhibit moderate selectivity for PTP1B over the closely related T-cell phosphatase. The molecular basis for the selectivity has been confirmed by single point mutation at position 52, where the two phosphatases differ by a phenylalanine-to-tyrosine switch. These compounds present a novel platform for the development of potent and selective PTP1B inhibitors.