Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4ß by µ-opioid receptors. ML204 inhibited TRPC4ß-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 µm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4ß currents activated through either µ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTP?S), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 µm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTP?S, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Maximising trace soil evidence: An improved recovery method developed during investigation of a $26 million bank robbery

Obtaining as much particulate material as possible from questioned items is desirable in forensic science as this allows a range of analyses to be undertaken and the retention of material for others to check. A method of maximising particulate recovery is described using a kidnap case, where minimal staining on clothing (socks) remained as possible indications of where the victim had been held captive. Police intelligence led to a hostage scene that was sampled. Brushing of the socks recovered about 50 sand grains with some silt: ultrasonic agitation and centrifuging recovered over 300 grains of sand, silt and clay. These were visually compared to scene and control samples, allowing exclusion of 52 samples and the retention of one comparison sample as well as other possibles, saving time and money, but maximising sample quantity and quality. © 2011 Elsevier Ireland Ltd.

Raman spectroscopic studies of the cure of dicyclopentadiene (DCPD)

The cure of polydicyclopentadiene conducted by ring-opening metathesis polymerisation in the presence of a Grubbs catalyst was studied using non-invasive Raman spectroscopy. The spectra of the monomer precursor and polymerised product were fully characterised and all stages of polymerisation monitored. Because of the monomer's high reactivity, the cure process is adaptable to reaction injection moulding and reactive rotational moulding. The viscosity of the dicyclopentadiene undergoes a rapid change at the beginning of the polymerisation process and it is critical that the induction time of the viscosity increase is determined and controlled for successful manufacturing. The results from this work show non-invasive Raman spectroscopic monitoring to be an effective method for monitoring the degree of cure, paving the way for possible implementation of the technique as a method of real-time analysis for control and optimisation during reactive processing. Agreement is shown between Raman measurements and ultrasonic time of flight data acquired during the initial induction period of the curing process. (c) 2004 Elsevier B.V. All rights reserved.

Hydrodynamic cavitation in micro channels with channel sizes of 100 and 750 micrometers

Decreasing the constriction size and residence time in hydrodynamic cavitation is predicted to give increased hot spot temperatures at bubble collapse and increased radical formation rate. Cavitation in a 100 x 100 mu m(2) rectangular micro channel and in a circular 750 mu m diameter milli channel has been investigated with computational fluid dynamics software and with imaging and radical production experiments. No radical production has been measured in the micro channel. This is probably because there is no spherically symmetrical collapse of the gas pockets in the channel which yield high hot spot temperatures. The potassium iodide oxidation yield in the presence of chlorohydrocarbons in the milli channel of up to 60 nM min(-1) is comparable to values reported on hydrodynamic cavitation in literature, but lower than values for ultrasonic cavitation. These small constrictions can create high apparent cavitation collapse frequencies.

Voltage-activated currents in cardiac myocytes of the blue mussel, Mytilus edulis

Voltage-sensitive ionic currents were identified and characterised in ventricular myocytes of the bivalve mollusc, Mytilus edulis, using the whole-cell patch-clamp technique. Two outward currents could be distinguished. A potassium A current (I-A) activated at - 30 mV from a holding potential of - 60 mV. This transient current was inactivated by holding the cells at a potential of - 40 mV and was also blocked by applying 4-aminopyridine (3 mM) to the external bath solution. A second current was identified as a delayed rectifier (I-K). This also activated at - 30 mV but exhibited a sustained time course and was still activated at a holding potential of - 40 mV. Both outward currents were reduced in the presence of tetraethylammonium ions (30 mM). A small number of heart cells also showed an inward sodium current (I-Na). This current appeared at potentials more positive than - 50 mV, reached a maximum at - 20 mV, and decreased with further depolarisation. I-Na was inactivated at a holding potential of - 40 mV and was blocked by tetrodotoxin (1 mu M). A second inward current had a sustained time course and was not inactivated by holding the cell at a potential of -40 mV, and was also not abolished by tetrodotoxin. This current peaked at 0 mV, decreasing with further depolarisation. Furthermore, it was enhanced by the addition of barium ions (3 mM) to the bath and was blocked by external cobalt (2 mM) or nifedipine (15 mu M) These findings are consistent with this being an L-type calcium current (I-Ca) The possible physiological roles of these currents in M. edulis heart are discussed. (C) 1999 Elsevier Science Inc. All rights reserved.

EFFECT OF ULTRASOUND ON THE KINETICS OF OXIDATION OF OCTAN-2-OL AND OTHER SECONDARY ALCOHOLS WITH SODIUM-BROMATE, MEDIATED BY RUTHENIUM TETRAOXIDE IN A BIPHASIC SYSTEM

The initial rate of oxidation of octan-2-ol and other secondary alcohols to their ketones with NaBrO3, mediated by RuO4 in an aqueous-CCl4 biphasic system, is greater with ultrasonic irradiation than by stirring alone. Under ultrasonic irradiation the initial rate of oxidation of octan-2-ol increases with increasing % duty cycle, [RuO4] and [NaBrO3]. The kinetics of alcohol oxidation appear to be closely linked with the oxidative dissolution of RuO2 to RuO4 by NaBrO3. The observed enhancement in rate with ultrasonic irradiation appear to be association, at least in part, with the increase in interfacial surface area via the formation of an emulsion of aqueous microdroplets containing NaBrO3 in the CCl4 layer containing the non-water-soluble secondary alcohol.

EFFECT OF ULTRASOUND ON THE KINETICS OF REDUCTION OF HEXACYANOFERRATE(III) BY THIOSULFATE IONS MEDIATED BY RUTHENIUM DIOXIDE HYDRATE

Ultrasound promotes the reduction of hexacyanoferrate(III) by thiosulfate ions mediated by RuO2 . xH(2)O under diffusion-controlled conditions. There is a strong correlation between the measured first-order rate constant and the absorbance of the dispersion, which, in turn, is closely related to the specific surface area of the catalyst. The enhancement in rate with ultrasonic irradiation appears to be largely associated with the dispersive action of the ultrasound on the aggregated particles of RuO2 . xH(2)O. The rate of reaction increases with increasing %duty cycle and ultrasonic intensity. The measured overall activation energies for the reaction with and without ultrasound, i.e. 18 +/- 1 and 20 +/- 1 kJ mol(-1), respectively, are very similar to those expected for a diffusion-controlled reaction. The homogeneous reaction is not promoted by ultrasound.

Textural, viscoelastic and mucoadhesive properties of pharmaceutical gels composed of cellulose polymers

This study examined the mechanical/textural, viscoeiastic and mucoadhesive properties of a range of aqueous gels composed of either hydroxyethylcellulose (HEC) or sodium carboxymethylcellulose (Na CMC). The mechanical/textural properties of each formulation were determined using texture profile analysis. The viscoelastic properties of each formulation were examined over a defined frequency range (0.01-1.0 Hz) using oscillatory rheometry in conjunction with stainless steel parallel plate geometry. The mucoadhesive properties of the gels were evaluated by measuring the tensile force required to overcome the gel/mucin adhesive interaction. Both gel hardness and compressibility, properties that affect the ease of product removal from a container and spreadability, increased as a function of increasing polymer concentrations. This is attributed to the effects of HEC and Na CMC on gel viscosity. Gel adhesiveness, a property related to bioadhesion, also increased as a function of polymer concentration and is attributed to the reported adhesive nature of these polymers. Increasing frequency of oscillation increased the storage and loss moduli yet decreased bath the dynamic viscosity of each gel type and also the loss tangent of HEC (but not Na CMC) gels. Therefore, following exposure to the range of oscillatory stresses that may be expected in vivo, HEC gels will be more susceptible than Na CMC gels to alterations in these rheological properties. Consequently, it would be expected that the clinical performance of HEC gels will be modified to a greater extent than Na CMC gels. In general, HEC gels exhibited a greater elastic nature than Na CMC gels over the frequency range employed for oscillation The storage and loss moduli and dynamic viscosity of both gel types increased, yet the loss tangent of both gel types decreased as a function of increasing polymer concentration. Gel mucoadhesive strength was dependent on both the time of contact of the formulation with mucin and also on polymer concentration. In conclusion, this study has characterised a number of gels containing either HEC or Na CMC in terms of their mechanical/textural, viscoelastic and mucoadhesive properties. Due to its relevance to the clinical performance, it is suggested that the information derived from these methods may be usefully combined to provide a more rational basis for the selection of polymers and their formulation as topical drug delivery systems. (C) 1997 Elsevier Science B.V.

Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

THE EFFECTS OF 3 NON-ANTIBIOTIC, ANTIMICROBIAL AGENTS ON THE SURFACE HYDROPHOBICITY OF CERTAIN MICROORGANISMS EVALUATED BY DIFFERENT METHODS

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.

Understanding the Behaviour of PET in Stretch Blow Moulding

This paper is concerned with understanding the behaviour of Polyethylene Terephthalate (PET) in the injection stretch blow moulding (ISBM) process where it is typically bi-axially stretched to form bottles for the packaging industry. Preforms which have been pre sprayed with a pattern and heated in an oil bath have been stretched and blown in free air using a lab scale ISBM machine whilst being monitored via high speed video. The images have subsequently been analysed using a digital image correlation system (VIC 3D). Results are presented showing the typical deformation modes and strain rates encountered in the ISBM process.

Statistical-mechanical description of classical test-particle dynamics in the presence of an external force field: modelling noise and damping from first principles

Aiming to establish a rigorous link between macroscopic random motion (described e.g. by Langevin-type theories) and microscopic dynamics, we have undertaken a kinetic-theoretical study of the dynamics of a classical test-particle weakly coupled to a large heat-bath in thermal equilibrium. Both subsystems are subject to an external force field. From the (time-non-local) generalized master equation a Fokker-Planck-type equation follows as a "quasi-Markovian" approximation. The kinetic operator thus defined is shown to be ill-defined; in specific, it does not preserve the positivity of the test-particle distribution function f(x, v; t). Adopting an alternative approach, previously introduced for quantum open systems, is proposed to lead to a correct kinetic operator, which yields all the expected properties. A set of explicit expressions for the diffusion and drift coefficients are obtained, allowing for modelling macroscopic diffusion and dynamical friction phenomena, in terms of an external field and intrinsic physical parameters.