Encoding combinatorial libraries: A novel application of fluorescent silica colloids

A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 mum silica colloidal particles (reporters) that contain specific and identifiable combinations of fluorescent byes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 +/- 23 in 2500 mum(2) area for PEI/PAA coated and 11 +/- 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 mum(2) area of green-labeled bead for PEI/PAA coated and 26 +/- 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the xi -potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 +/- 16 in 2500 mum(2) area for PDADMAC/PSSS and 54 +/- 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using gamma irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.

Quantum measurement and stochastic processes in mesoscopic conductors

In quantum measurement theory it is necessary to show how a, quantum source conditions a classical stochastic record of measured results. We discuss mesoscopic conductance using quantum stochastic calculus to elucidate the quantum nature of the measurement taking place in these systems. To illustrate the method we derive the current fluctuations in a two terminal mesoscopic circuit with two tunnel barriers containing a single quasi bound state on the well. The method enables us to focus on either the incoming/ outgoing Fermi fields in the leads, or on the irreversible dynamics of the well state itself. We show an equivalence between the approach of Buttiker and the Fermi quantum stochastic calculus for mesoscopic systems.

Modelling solute transport in structured soils: Performance evaluation of the ADR and TRM models

The movement of chemicals through the soil to the groundwater or discharged to surface waters represents a degradation of these resources. In many cases, serious human and stock health implications are associated with this form of pollution. The chemicals of interest include nutrients, pesticides, salts, and industrial wastes. Recent studies have shown that current models and methods do not adequately describe the leaching of nutrients through soil, often underestimating the risk of groundwater contamination by surface-applied chemicals, and overestimating the concentration of resident solutes. This inaccuracy results primarily from ignoring soil structure and nonequilibrium between soil constituents, water, and solutes. A multiple sample percolation system (MSPS), consisting of 25 individual collection wells, was constructed to study the effects of localized soil heterogeneities on the transport of nutrients (NO3-, Cl-, PO43-) in the vadose zone of an agricultural soil predominantly dominated by clay. Very significant variations in drainage patterns across a small spatial scale were observed tone-way ANOVA, p < 0.001) indicating considerable heterogeneity in water flow patterns and nutrient leaching. Using data collected from the multiple sample percolation experiments, this paper compares the performance of two mathematical models for predicting solute transport, the advective-dispersion model with a reaction term (ADR), and a two-region preferential flow model (TRM) suitable for modelling nonequilibrium transport. These results have implications for modelling solute transport and predicting nutrient loading on a larger scale. (C) 2001 Elsevier Science Ltd. All rights reserved.

Physiological role of calcium-activated potassium currents in the rat lateral amygdala

Principal neurons in the lateral nucleus of the amygdala (LA) exhibit a continuum of firing properties in response to prolonged current injections ranging from those that accommodate fully to those that fire repetitively. In most cells, trains of action potentials are followed by a slow after hyperpolarization (AHP) lasting several seconds. Reducing calcium influx either by lowering concentrations of extracellular calcium or by applying nickel abolished the AHP, confirming it is mediated by calcium influx. Blockade of large conductance calcium-activated potassium channel (BK) channels with paxilline, iberiotoxin, or TEA revealed that BK channels are involved in action potential repolarization but only make a small contribution to the fast AHP that follows action potentials. The fast AHP was, however, markedly reduced by low concentrations of 4-aminopyridine and alpha-dendrotoxin, indicating the involvement of voltage-gated potassium channels in the fast AHP. The medium AHP was blocked by apamin and UCL1848, indicating it was mediated by small conductance calcium-activated potassium channel (SK) channels. Blockade of these channels had no effect on instantaneous firing. However, enhancement of the SK-mediated current by 1-ethyl-2-benzimidazolinone or paxilline increased the early interspike interval, showing that under physiological conditions activation of SK channels is insufficient to control firing frequency. The slow AHP, mediated by non-SK BK channels, was apamin-insensitive but was modulated by carbachol and noradrenaline. Tetanic stimulation of cholinergic afferents to the LA depressed the slow AHP and led to an increase in firing. These results show that BK, SK, and non-BK SK-mediated calcium-activated potassium currents are present in principal LA neurons and play distinct physiological roles.

Channels underlying neuronal calcium-activated potassium currents

In many cell types rises in cytosolic calcium, either due to influx from the extracellular space, or by release from an intracellular store activates calcium dependent potassium currents on the plasmalemma. In neurons, these currents are largely activated following calcium influx via voltage gated calcium channels active during the action potentials. Three types of these currents are known: I-c. I-AHP and I-sAHP. These currents can be distinguished by clear differences in their pharmacology and kinetics. Activation of these potassium currents modulates action potential time course and the repetitive firing properties of neurons. Single channel studies have identified two types of calcium-activated potassium channel which can also be separated on biophysical and pharmacological grounds and have been named BK and SK channels. It is now clear that BK channels underlie Ic whereas SK channels underlie I-AHP. The identity of the channels underlying I-sAHP are not known. In this review, we discuss the properties of the different types of calcium-activated potassium channels and the relationship between these channels and the macroscopic currents present in neurons. (C) 2002 Elsevier Science Ltd. All rights reserved.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6’) common to both the glycine receptor (GlyR) and GABAA receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter and the main pore blocker binding site. Recent studies have suggested that the GlyRs and GABAARs have divergent open state pore structures at the 6’ position. When both the human a1T6’C homomeric GlyR and the rat a1T6’Cb1T6’C heteromeric GABAAR were expressed in HEK293 cells, their 6’ residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidising agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABAAR pore structure at the 6’ level may vary between different expression systems.

Discriminating between task-relevant and task-irrelevant stimuli - The effects on autonomic orienting and secondary task reaction time

The effect that the difficulty of the discrimination between task-relevant and task-irrelevant stimuli has on the relationship between skin conductance orienting and secondary task reaction time (RT) was examined. Participants (N = 72) counted the number of longer-than-usual presentations of one shape (task-relevant) and ignored presentations of another shape (task-irrelevant). The difficulty of discriminating between the two shapes varied across three groups (low, medium, and high difficulty). Simultaneous with the primary counting task, participants performed a secondary RT task to acoustic probes presented 50, 150, and 2000 ms following shape onset. Skin conductance orienting was larger, and secondary RT at the 2000 ms probe position was slower during task-relevant shapes than during task-irrelevant shapes in the low-difficulty group. This difference declined as the discrimination difficulty was increased, such that there was no difference in the high-difficulty group. Secondary RT was slower during task-irrelevant shapes than during task-relevant shapes only in the medium-difficulty group-and only at the 150 ms probe position in the first half of the experiment. The close relationship between autonomic orienting and secondary RT at the 2000 ms probe position suggests that orienting reflects the resource allocation that results from the number of matching features between a stimulus input and a mental representation primed as significant.

Embracing ligands. A synthetic strategy towards new nitrogen-thioether multidentate ligands and characterization of the cobalt(III) complexes

The synthesis of the hexadentate ligand 2,2,9,9-tetra(methyleneamine)-4,7-dithiadecane (EtN(4)S(2)amp) is reported. The ligand is of a type in which bifurcations of the chain occur at atoms other than donor atoms. The cobalt(III) complex [Co(EtN(4)S(2)amp)](3+) (1) was isolated and characterized. The synthetic methodology also results in a number of by-products, notably 2,9,9-tris(methyleneamine)-9-methylenehydroxy-4,7-dithiadecane (Et(HO)N(3)S(2)amp) and an eleven-membered pendant arm macrocyclic ligand 6,10-dimethyl-6,10-bis(methyleneamine)-1,4-dithia-8-azaacycloundec-7- ene (dmatue). The complexes [Co(Et(HO)N(3)S(2)amp)](3+) (2), in which the alcohol is coordinated to the metal ion, and [Co(dmatue)Cl](2+) (4) were isolated and characterized. Et(HO)N(3)S(2)amp also undergoes complexation with cobalt(III) to produce two isomers endo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (endo-3) and exo-[Co(Et(HO) N(3)S(2)amp)Cl](2+) (exo-3), both with an uncoordinated alcohol group. endo- 3 has the alcohol positioned cis, and exo-3 trans, to the sixth metal coordination site. Reaction of 1 with isobutyraldehyde, paraformaldehyde and base in dimethylformamide results in the encapsulated complex [Co(1,5,5,9,13,13-hexamethyl-18,21-dithia-3,7,11,15-tetraazabicyclo[7.7.6]docosa- 3,14-diene)](ClO4)(3) . 2H(2)O ([Co(Me(6)docosadieneN(4)S(2))](3+) ( 5). All complexes have been characterized by single crystal X-ray study. The low-temperature (11 K) absorption spectrum of 1 has been measured in Nafion films with spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands observed. The octahedral ligand-field parameters were determined (10Dq = 22570 cm(-1), B = 551 cm(-1); C = 3500 cm(-1)). For 5 10Dq and B were determined (20580 cm(-1); 516 cm(-1), respectively) and compared with those for similar expanded cavity complexes [Co(Me(8)tricosatrieneN(6))](3+) and [Co(Me(5)tricosatrieneN(6))](3+).

Atmospheric corrosion of copper at Heron Island

Atmospheric corrosion tests have been conducted at Heron Island, Queensland, a low SO2 marine environment. The corrosion rates for copper followed the linear bi-logarithmic law. X-ray diffraction identified atacamite (basic copper chloride) and cuprite (cuprous oxide) as the predominant corrosion products. Biogenic and anthropogenic sources of pollution did not have a measurable influence.

Odorant-induced currents in intact patches from rat olfactory receptor neurons: Theory and experiment

Odorant-induced currents in mammalian olfactory receptor neurons have proved difficult to obtain reliably using conventional whole-cell recording. By using a mathematical model of the electrical circuit of the patch and rest-of-cell, we demonstrate how cell-attached patch measurements can be used to quantitatively analyze responses to odorants or a high (100 mM) K+ solution. High K+ induced an immediate current flux from cell to pipette, which was modeled as a depolarization of similar to 52 mV, close to that expected from the Nernst equation (56 mV), and no change in the patch conductance. By contrast, a cocktail of cAMP-stimulating odorants induced a current flux from pipette into cell following a significant (4-10 s) delay. This was modeled as an average patch conductance increase of 36 pS and a depolarization of 13 mV, Odorant-induced single channels had a conductance of 16 pS. In cells bathed with no Mg2+ and 0.25 mM Ca2+, odorants induced a current flow from cell to pipette, which was modeled as a patch conductance increase of similar to 115 pS and depolarization of similar to 32 mV, All these results are consistent with cAMP-gated cation channels dominating the odorant response, This approach, which provides useful estimates of odorant-induced voltage and conductance changes, is applicable to similar measurements in any small cells.

The glycine receptor

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. The GlyR comprises a pentameric complex that forms a chloride-selective transmembrane channel, which is predominantly expressed in the spinal cord and brain stem. We review the pharmacological and physiological properties of the GlyR and relate this information to more recent insights that have been obtained through the cloning and recombinant expression of the GlyR subunits. We also discuss insights into our understanding of GlyR structure and function that have been obtained by the genetic characterisation of various heritable disorders of glycinergic neurotransmission. (C) 1997 Elsevier Science Inc.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [S-35]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximate to 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane off. coli (1) and therefore may have profound implication for the activation and/or ''multimerization'' of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium. (C) 1997 Academic Press.

The effect of emotional and attentional processes on blink startle modulation and on electrodermal responses

Emotional accounts of startle modulation predict that startle is facilitated if elicited during aversive foreground stimuli. Attentional accounts hold that startle is enhanced if startle-eliciting stimulus and foreground stimulus are in the same modality. Visual and acoustic foreground stimuli and acoustic startle probes were employed in aversive differential conditioning and in a stimulus discrimination task. Differential conditioning was evident in electrodermal responses and blink latency shortening in both modalities, but effects on magnitude facilitation were found only for visual stimuli. In the discrimination task, skin conductance responses, blink latency shortening, and blink magnitude facilitation were larger during to-be-attended stimuli regardless of stimulus modality. The present results support the notion that attention and emotion can affect blink startle modulation during foreground stimuli.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80 degrees C) or acyl chloride (in the presence of pyridine at 0 degrees C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend, The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length. (C) 1997 Elsevier Science B.V.