A Novel Selective Muscarinic Acetylcholine Receptor Subtype 1 Antagonist Reduces Seizures without Impairing Hippocampus-Dependent Learning

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M 1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M-1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)benzo[c][1,2,5]thiadiazole-4-sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M-1 mAChRs relative to M-2-M-5. Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M-1 mAChRs, a surprising finding given the high level of M-1 mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-D-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilo-carpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M-1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.

Non-linear finite-element analysis of punching capacities of steel-concrete bridge deck slabs

Punching failure is the common failure mode in concrete bridge deck slabs when these structural components are subjected to local patch loads, such as tyre loads. Past research has shown that reinforced concrete slabs in girder–slab type bridges have a load-carrying capacity far greater than the ultimate static loads predicted by traditional design methods, because of the presence of compressive membrane action. However, due to the instability problems from punching failure, it is difficult to predict ultimate capacities accurately in numerical analyses. In order to overcome the instability problems, this paper establishes an efficient non-linear finite-element analysis using the commercial finite-element package Abaqus. In the non-linear finite-element analysis, stabilisation methods were adopted and failure criteria were established to predict the ultimate punching behaviour of deck slabs in composite steel–concrete bridges. The proposed non-linear finite-element analysis predictions showed a good correlation on punching capacities with experimental tests.

Effect of glucose on endothelin-1-induced calcium transients in cultured bovine retinal pericytes

Published work has shown that endothelin-l-induced contractility of bovine retinal pericytes is reduced after culture in high concentrations of glucose. The purpose of the present study was to establish the profile of endothelin-l-induced calcium transients in pericytes and to identify changes occurring after culture in high concentrations of glucose. Glucose had no effect on basal levels of cytosolic calcium or on endothelin-l-induced calcium release from intracellular stores. However, influx of calcium from the extracellular medium after endothelin-l stimulation was reduced in pericytes that had been cultured in 25 mM D-glucose. L-type Ca2+ currents were identified by patch clamping. The L-type Ca2+ channel agonist, (-)-Bay K8644, caused less influx of calcium from the extracellular medium in pericytes that had been cultured in 25 mM D-glucose than in those cultured with 5 mM D-glucose. However, 3-O-methylglucose, a nonmetabolizable analogue of glucose which can cause glycation, had similar effects to those of high concentrations of glucose. The results suggest that reduced function of the L-type Ca2+ channel that occurs in bovine retinal pericytes after culture in high concentrations of D-glucose is probably due to glycation of a channel protein.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

How do grazers achieve their distribution? A continuum of models from random diffusion to the ideal free distribution using biased random walks

A conceptual model is described for generating distributions of grazing animals, according to their searching behavior, to investigate the mechanisms animals may use to achieve their distributions. The model simulates behaviors ranging from random diffusion, through taxis and cognitively aided navigation (i.e., using memory), to the optimization extreme of the Ideal Free Distribution. These behaviors are generated from simulation of biased diffusion that operates at multiple scales simultaneously, formalizing ideas of multiple-scale foraging behavior. It uses probabilistic bias to represent decisions, allowing multiple search goals to be combined (e.g., foraging and social goals) and the representation of suboptimal behavior. By allowing bias to arise at multiple scales within the environment, each weighted relative to the others, the model can represent different scales of simultaneous decision-making and scale-dependent behavior. The model also allows different constraints to be applied to the animal's ability (e.g., applying food-patch accessibility and information limits). Simulations show that foraging-decision randomness and spatial scale of decision bias have potentially profound effects on both animal intake rate and the distribution of resources in the environment. Spatial variograms show that foraging strategies can differentially change the spatial pattern of resource abundance in the environment to one characteristic of the foraging strategy.</

Animal foraging from an individual perspective: an object orientated model

A model system, HOOFS (Hierarchical Object Orientated Foraging Simulator), has been developed to study foraging by animals in a complex environment. The model is implemented using an individual-based object-orientated structure. Different species of animals inherit their general properties from a generic animal object which inherits from the basic dynamic object class. Each dynamic object is a separate program thread under the control of a central scheduler. The environment is described as a map of small hexagonal patches, each with their own level of resources and a patch-specific rate of resource replenishment. Each group of seven patches (0th order) is grouped into a Ist order super-patch with seven nth order super-patches making up a n + 1th order super-patch for n up to a specified value. At any time each animal is associated with a single patch. Patch choice is made by combining the information on the resources available within different order patches and super-patches along with information on the spatial location of other animals. The degree of sociality of an animal is defined in terms of optimal spacing from other animals and by the weighting of patch choice based on social factors relative to that based on food availability. Information, available to each animal, about patch resources diminishes with distance from that patch. The model has been used to demonstrate that social interactions can constrain patch choice and result in a short-term reduction of intake and a greater degree of variability in the level of resources in patches. We used the model to show that the effect of this variability on the animal's intake depends on the pattern of patch replenishment. (C) 1998 Elsevier Science B.V. All rights reserved.</p>

Beyond the ideal free distribution: More general models of predator distribution

The ideal free distribution model which relates the spatial distribution of mobile consumers to that of their resource is shown to be a limiting case of a more general model which we develop using simple concepts of diffusion. We show how the ideal free distribution model can be derived from a more general model and extended by incorporating simple models of social influences on predator spacing. First, a free distribution model based on patch switching rules, with a power-law interference term, which represents instantaneous biased diffusion is derived. A social bias term is then introduced to represent the effect of predator aggregation on predator fitness, separate from any effects which act through intake rate. The social bias term is expanded to express an optimum spacing for predators and example solutions of the resulting biased diffusion models are shown. The model demonstrates how an empirical interference coefficient, derived from measurements of predator and prey densities, may include factors expressing the impact of social spacing behaviour on fitness. We conclude that empirical values of log predator/log prey ratio may contain information about more than the relationship between consumer and resource densities. Unlike many previous models, the model shown here applies to conditions without continual input. (C) 1997 Academic Press Limited.</p>

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

Coelum non animum muto? Milton's Neo-Latin Poetry and Catholic Italy

Milton’s Elegiarum Liber, the first half of his Poemata published in Poems of Mr John Milton Both English and Latin (1645), concludes with a series of eight Latin epigrams: five bitterly anti-Catholic pieces on the failed Gunpowder Plot of 1605, followed by three encomiastic poems hymning the praises of an Italian soprano, Leonora Baroni, singing in Catholic Rome. The disparity in terms of subject matter and tone is self-evident yet surprising in an epigrammatic series that runs sequentially. Whereas the gunpowder epigrams denigrate Rome, the Leonora epigrams present the city as a cultured hub of inclusivity, the welcome host of a Neapolitan soprano. In providing the setting for a human song that both enthrals its audience and attests to the presence of a divine power, Rome now epitomizes something other than brute idolatry, clerical habit or doctrine. And for the poet this facilitates an interrogation of theological (especially Catholic) doctrines. Coelum non animum muto, dum trans mare curro wrote the homeward-bound Milton in the autograph book of Camillo Cardoini at Geneva on 10 June 1639. But that this was an animus that could indeed acclimatize to religious and cultural difference is suggested by the Latin poems which Milton “patch [ed] up” in the course of his Italian journey. Central to that acclimatisation, as this chapter argues, is Milton’s quasi-Catholic self-fashioning. Thus Mansus offers a poetic autobiography of sorts, a self-inscribed vita coloured by intertextually kaleidoscopic links with two Catholic poets of Renaissance Italy and their patron; Ad Leonoram 1 both invokes and interrogates Catholic doctrine before a Catholic audience only to view the whole through the lens of a neo-Platonic hermeticism that may refreshingly transcend religious difference. Finally, Epitaphium Damonis, composed upon Milton’s return home, seems to highlight the potential interconnectedness of Protestant England and Catholic Italy, through the Anglo-Italian identity of its deceased subject, and through a pseudo-monasticism suggested by the poem’s possible engagement with the hagiography of a Catholic Saint. Perhaps continental travel and the physical encounter with the symbols, personages and institutions of the other have engendered in the Milton of the Italian journey a tolerance or, more accurately, the manipulation of a seeming tolerance to serve poetic and cultural ends.


First reviewer:
Haan: a fine piece by the senior neo-Latinist in Milton studies.

Second reviewer:
Chapter 7 is ... a high-spot of the collection. Its argument that in his Latin poetry Milton’s is a ‘quasi-Catholic self-fashioning’ stressing ‘the potential interconnectedness of Protestant England and Catholic Italy’ is striking and is advanced with learning, clarity and insight. Its sensitive exploration of the paradox of Milton’s coupling of humanistically complimentary and tolerant address to Roman Catholic friends with fiercely Protestant partisanship demonstrates that there is much greater complexity to his poetic persona than the self-construction and self-presentation of the later works would suggest. The essay is always adroit and sure-footed, often critically acute and illuminating (as, for example, in its discussion of the adjective and adverb mollis and molliter in Mansus, or in the identification in n. 99 of hitherto unnoticed Virgilian echoes). It has the added merits of being very well written, precise and apt in its citation of evidence, and absolutely central to the concerns of the volume.

Hydrogel-Forming Microneedle Arrays for Enhanced Transdermal Drug Delivery

Unique microneedle arrays prepared from crosslinked polymers, which contain no drug themselves, are described. They rapidly take up skin interstitial fluid upon skin insertion to form continuous, unblockable, hydrogel conduits from attached patch-type drug reservoirs to the dermal microcirculation. Importantly, such microneedles, which can be fabricated in a wide range of patch sizes and microneedle geometries, can be easily sterilized, resist hole closure while in place, and are removed completely intact from the skin. Delivery of macromolecules is no longer limited to what can be loaded into the microneedles themselves and transdermal drug delivery is now controlled by the crosslink density of the hydrogel system rather than the stratum corneum, while electrically modulated delivery is also a unique feature. This technology has the potential to overcome the limitations of conventional microneedle designs and greatly increase the range of the type of drug that is deliverable transdermally, with ensuing benefits for industry, healthcare providers and, ultimately, patients.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Contribution of reverse Na+/Ca2+ exchange to spontaneous activity in interstitial cells of cajal in the rabbit urethra

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

A randomised double-blind placebo-controlled phase II study of AGI004 for control of chemotherapy-induced diarrhoea

Background: AGI004 is a controlled-release transdermal patch preparation of mecamylamine. We conducted a randomised placebo-controlled phase II study of two dose levels of AGI004 in chemotherapy-induced diarrhoea (CID).

Methods: Adult patients receiving chemotherapy who had experienced diarrhoea (NCI grade 1-2) during previous cycles of chemotherapy were eligible. In all, 64 patients were randomised to receive AGI004 4mg then 8mg per 24 h transdermal patch or placebo for two sequential cycles of chemotherapy. Patients' severity of diarrhoea was physician-assessed using NCI grade of diarrhoea and patient-assessed using information recorded in daily diaries of bowel movements.

Results: Overall AGI004 doubled the odds of a response to treatment on the first day of chemotherapy based on physician assessment of NCI grade of diarrhoea compared with placebo (odds ratio = 2.0, 90% confidence interval: 0.9-4.5) and there was a trend to improved response rates for AGI004 for the full treatment cycle although these results were not statistically significant. There was also evidence of significantly improved response rates based on patient assessment of diarrhoea both overall (P = 0.05) and at the 8-mg dose level (P = 0.02) compared with placebo.

Conclusion: AGI004 demonstrated effectiveness in reducing chemotherapy-associated diarrhoea, with results suggesting response across multiple measurements of diarrhoea. Treatment was well tolerated with no drug-related adverse events. Further evaluation of this agent in the management of CID is warranted.