John Heptinstall

The endogenous production of hydrogen sulphide in intrauterine tissues

BackgroundHydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.MethodsThe production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated.ResultsThe order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion.ConclusionRat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy.

A Magneto-Optic Route toward the In Vivo Diagnosis of Malaria: Preliminary Results and Preclinical Trial Data

We report the development of magneto-optic technology for the rapid quantitative diagnosis of malaria that may also be realizable in a noninvasive format. Hemozoin, the waste product of malarial parasitic action on hemoglobin, is produced in a form that under the action of an applied magnetic field gives rise to an induced optical dichroism characteristic of the hemozoin concentration. Here we show that precise measurement of this induced dichroism may be used to determine the level of malarial infection because this correlates, albeit in a complex manner throughout the infection cycle, with the concentration of hemozoin in the blood and tissues of infected patients. Under conservative assumptions for the production of hemozoin as a function of parasitemia, initial results indicate that the technique can match or exceed other current diagnostic techniques. The validity of the approach is confirmed by a small preliminary clinical trial on 13 patients, and measurements on live parasitized cells obtained from in vitro culture verify the possibility of producing in vivo diagnostic instrumentation.

Improved gel-protected bilayers

We report improved techniques for the fabrication of biomimetic membranes protectively sandwiched between two slabs of agarose gel and the evaluation of their characteristics relevant for use in a biosensor based on gated channel proteins. The gel protects the membrane from mechanical contact, from low-energy solvents and from drying out. The new methods allow the preparation in high yield of sensor cells with a front gel thickness less than 100 μm. During fabrication, gel-protected membranes of DOPC (dioleoyl phosphatidylcholine) readily incorporate valinomycin in functional form. DOPC membranes respond as expected and within a few minutes to aqueous solutions of gramicidin applied to the front gel layer. In long-term studies of protected membranes of the synthetic lipid PSPC, no failures were observed over runs of up to 3 weeks and the membrane leakage decreased, reaching within 7 days values comparable to those reported for unsupported bilayer lipid membranes. Initial attempts to incorporate functional nicotinic acetylcholine receptors have shown promise.

Sonoelectrochemical effects in electro-organic systems.

This paper describes recent studies in organic sonoelectrochemistry at Coventry University, including the oxidation of thiophene monoxides, degradation of dye pollutants, formation of conducting polymers and electrosynthetic modification of proteins.

A robust gel–bilayer channel biosensor

We demonstrate a novel protective configuration for a gated channel biosensor. The bilayer membrane containing the channel proteins is formed by a simple self-assembly technique ensuring continuous coverage of the interface between two slabs of agarose gel by a biomimetic lamella in a fluid state. The gel protects both membrane surfaces from mechanical shock and contact with low-energy media while allowing diffusion of biomolecules up to 10 MDa in weight. The technique has been demonstrated using both dioleoyl-phosphatidylcholine (DOPC) and a phosphatidylcholine lipid cross-linked with a short polysiloxane chain (PSPC). The conductance per unit area of the channel-free membrane produced by this method was less than 25 S m−2 for DOPC and 2 S m−2 for PSPC, and the bilayer nature of the barrier in both cases has been demonstrated by measurement of the capacitance. The applicability to sensors has been confirmed using gramicidin-D, a 1·1 kDa unilamellar lipid bilayer pore former, and partially confirmed using valinomycin, a selective ion transporter. On incorporation of gramicidin the membrane conductance increased by over an order of magnitude.

Electrosynthetic modification of proteins : electrooxidations at methionine and tryptophan in hen egg-white lysozyme

Electrosynthesis provides a novel methodology to produce specific and selective chemical reaction in proteins. Thus electrooxidation of hen egg-white lysozyme (HEWL) at + 1.2 V (vs sce) at a carbon anode in mildly acid buffer produces selective oxidation at methionine-105; while by increase in anodic potential, or by addition of acetonitrile co-solvent, subsequent oxidation at methionine-12 is observed. Further increase of potential in otherwise similar conditions additionally produces a novel cleavage between tryptophan-62 and tryptophan-63, the latter remaining intact and becoming the new N-terminus of the fragment. No reaction occurs at other tryptophan residues in HEWL, and histone H4, which contains no tryptophan residues, does not cleave in these conditions. The previously reported selective nitration at tyrosine residues by use of copper electrodes in alkaline medium is not observed in the acid electrolysis system and shows the fine control over protein modification available by manipulation of electrosynthetic parameters. The methodology offers wide implications.

The isomerisation of meso-amine substituted heptamethine dyes

From investigations into the absorption and fluorescence characteristics of several new meso-amine substituted heptamethine cyanine dyes and the commercially available laser dyes IR140, IR132 and IR144, it appears that these dyes exist in at least two interconvertible ground state isomeric conformations in organic solvents. From the position and nature of absorption and fluorescence characteristics, it is concluded that these conformers differ in the contribution to the chromophore of the meso-amine substituent, which either acts as an electron-donor by resonance or else withdraws by induction. Fluorescence emission below 900 nm can only be attributed to the conformer in which the meso-amine groups acts as an electron-donor by resonance. This gives rise to a system that is analogous to the allopolar isomerism exhibited by holomeropolar cyanine dyes.

Electrochemical modification of proteins. A review.

INTRODUCTION The majority of electrochemical studies of biomolecules have been analytical in nature, usually involving electron transfer by redox proteins. Synthetic applications of electrochemistry to proteins, leading to the production of recoverable products, has been far less widely studied. However, the use of proteins in electrosynthesis, exemplified by hen egg-white lysozyme, demonstrates that the reaction parameters of pH, potential difference, electrode material etc., can be varied in order to target different individual amino acid residues including tyrosine, tryptophan and methionine. This methodology provides a novel route to quantities of selectively-modified proteins.

Hydrolytic breakdown of meso-diphenylamine substituted heptamethine dye IR140 and isomerism of the merocyanine breakdown product

Abstract Nucleophilic attack by hydroxyl ions is found to occur preferentially at the C 2 position of the benzothiazolium heterocyclic moiety of the laser dye IR140, and not at the eneamine functionality that consists of the mesocarbon atom and the adjacent diphenylamine moiety, which is the other electrophilic site on the dye. The temperature-dependence of the absorption spectrum of the merocyanine breakdown product suggests that similar ground state isomerism is present in the breakdown product, as is found in the initial dye IR140. It is concluded that the non-fluorescent majority conformer of the merocyanine breakdown product is the one in which the meso-amine substituent is twisted so as to withdraw electron density by induction, and that the fluorescent minority conformer is that in which themeso-amine substituent is conjugated to act as an electron donor by resonance.

Electrooxidative nitration of phenols at copper electrodes

p-Cresol, as a small-molecule model for tyrosine residues in proteins, undergoes electrooxidative nitration in the presence of a nitrogen source, for example ammonia, in mildly alkaline aqueous solution at potentials in the range 0.8 to 1.4 V (vs sce). Anodized copper is the best electrode material of those studied and nitrogen sources in the increasing order of effectiveness are amides < amines ≅ proteins < ammonia, the latter giving a total of nitrocresols of ~ 30% from an initial p-cresol concentration of 0.5 mM. Azulene also nitrates in these conditions, but phenylethers (4-methyl methoxybenzene and 1,2-dimethoxy benzene) do not. The protein hen egg-white lysozyme (HEWL), in the absence of any other nitrogenous species, acts as a source of nitrating agent in the electrooxidative nitration of p-cresol thus substantiating our earlier finding of selective tyrosine nitration in proteins in the absence of any other nitrogen source. This electronitration reaction, unique in that there is no NO bond in any initial species, provides a novel and environmentally friendly route in mild conditions and is of particular benefit in the selective covalent modification of proteins.