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Biomedical subjects

M N Jones

Publications and source records attributed to M N Jones.

At least 19 recordsLinked to original sources
PubMed1992

Freeze-thaw effects on the detection of blood group substances in detergent extracts by enzyme-linked immunosorbent assays (ELISA).

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for human blood group antigens extracted from blood stains with the surfactant, n-octyl-beta-D-glucopyranoside (OBG), at concentrations below the critical micelle concentration can be increased by the introduction of a single freeze-thaw step. The ELISA signals increase from 3- to 4-fold for OBG extracts of 80 nl bloodstains. The ELISA signal enhancement occurs irrespective of the age of the bloodstains, at least for bloodstains up to 1 year old. The origin of the effect has been investigated and its is demonstrated that the freeze-thawing cycle increases the extent of adsorption of the blood group determinants in OBG-solubilized complexes onto microtitre plates. Gel filtration has been used to analyse the composition of OBG extracts of bloodstains in terms of the carriers of the blood group substances, protein and phospholipid in fresh and freeze-thawed extracts. It was found that freeze-thawing alters the distribution of blood group active material in the lipid-protein OBG complexes leading to a greater proportion of blood group active material in higher molecular weight complexes. The freeze-thaw effect is eliminated on the addition of a cryoprotectant, such as glycerol, and the factors which contribute to changes in the microstructure of OBG extracts on freeze-thawing are discussed.

Blood Group Antigens↗
PubMed1992

The thermal behaviour of phosphatidylcholine-glycophorin monolayers in relation to monolayer and bilayer internal pressure.

A thermodynamic relationship which allows calculation of the internal pressure (Pi) of a monolayer has been derived, viz: Pi = T(delta pi/delta T)A-pi. Surface pressure (pi)-area (A) isotherms were determined for dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and a mixture of the latter with 0.28 mol% glycophorin, an intrinsic membrane protein. The isotherms (10 degrees C-42 degrees C) were used to obtain values of (delta pi/delta T)A. Under conditions in which a monolayer is believed to most closely model a bilayer membrane (i.e. 37 degrees C and A = 0.6 nm2/molecule) internal pressures were: DOPC = 0.32 N m-1; DPPC = 0.13 N m-1 and glycophorin/DPPC = 0.36 N m-1. The results do not support some measurements on amphiphile solubility in natural membranes which had been interpreted as evidence of a large increase in internal pressure, due to the intrinsic membrane proteins.

Erythrocyte Membrane↗
PubMed1992

The effects of surfactants on the permeability of isolated perfused fish gills to urea.

1. The diffusional transfer capacity of [14C]urea in isolated perfused trout (Oncorhynchus mykiss) gills in the presence of sodium n-dodecylsulphate (SDS), n-dodecyltrimethylammonium bromide (DTAB) and p-t-octylbenzene oxyethylene10 (Triton X-100) has been measured over a range of surfactant concentrations. 2. Urea has been shown to be transported transcellularly through the respiratory cells of the gill secondary lamellae by passive diffusion. Each surfactant was found to markedly increase the rate of diffusion and the diffusional transfer capacity reached a steady-state at a particular surfactant concentration. 3. The steady state flux was increased by surfactant in the sequence DTAB greater than SDS greater than Triton X-100 and the surfactant concentrations in terms of the critical micelle concentration (CMC) at which the diffusional transfer capacities reached limiting values were 0.92 x CMC (SDS), 0.53 x CMC (DTAB) and 2.5 x CMC (Triton X-100). 4. Compared to interactions between isolated epithelial cells and the surfactants, the rates at which the surfactants changed the urea flux were slow, suggesting that the mucus layer plays a significant role in protecting the epithelial cells of the secondary lamellae from disruption. 5. Relative to the other surfactants, DTAB had the most marked effect on both the rate of flux change and on the magnitude of the change, at concentrations which are low relative to the CMC, suggesting a more specific interaction with the negatively charged mucus layer consistent with the toxic effects of quaternary ammonium compounds on aquatic organisms.

Animals↗
PubMed1992

Bacillus anthracis but not always anthrax.

Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B. cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B. anthracis which lack the plasmid carrying the capsule gene (pXO2). While these techniques cannot, of course, be used to confirm the identities of strains resembling B. anthracis but which also lack the plasmid carrying the toxin genes (pXO1), the likelihood that these also are bona fide B. anthracis becomes more acceptable. (As yet no naturally occurring pXO1-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

Animals↗
PubMed1991

A liposomal enzyme electrode for measuring glucose.

Enzyme electrodes have been described for measuring glucose but have been limited by the saturation kinetics of the glucose oxidase not allowing clinically relevant glucose concentrations to be measured (0-25 mM). One way of alleviating this problem is to use diffusion-controlled membranes which result in the enzyme experiencing a smaller substrate concentration than that of the bulk solution. As an extension of this concept we have encapsulated glucose oxidase in liposomes whereby the lipid bilayer wall provides the diffusion-limiting membrane as well as providing a biocompatible layer which is of particular relevance when blood glucose is to be measured. Linear ranges were found to embrace the required glucose concentrations and moreover by using liposomes prepared from different lipids, e.g., dimyristoyl (14:0) phosphatidylcholine (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC) and distearoyl (18:0) phosphatidylcholine (DSPC), the electrode response was shown to depend on the bilayer permeabilities in relation to the lipid phase transition temperatures and as a consequence the linear ranges were duly altered.

Biocompatible Materials↗
PubMed1991

The effect of blood serum on the size and stability of phospholipid liposomes.

Liposomes have been prepared by sonication (SUV) and reverse phase evaporation (REV) from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI), stearylamine and cholesterol. The effect of rat and human blood serum on the liposomes has been investigated by measurement of the particle size in the serum-liposome mixtures by photon correlation spectroscopy in the range of serum protein concentration up to approx. 25 mg ml-1. At low serum protein concentrations the measured particle sizes exceed those calculated from the known sizes and concentrations of liposomes and serum particles in the mixtures: a result consistent with serum-induced aggregation of the liposomes, but the aggregates dissociate at higher serum protein concentration. The effect of serum on the release of encapsulated [14C]glucose from REV liposomes has been investigated over a range of serum protein concentration by gel filtration. At low serum concentration a proportion of the liposomes remain intact but as the serum concentration is increased the size of the liposomes decreases with concomitant release of encapsulated glucose. At high serum concentrations (approx. 24 mg protein per ml) the larger liposomes in the distribution are disrupted and some of the liposomal lipid becomes associated with serum protein. The results are discussed with reference to the effect of blood on the uptake of liposomes by rat liver.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

The effect of blood on the uptake of liposomal lipid by perfused rat liver.

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) and stearylamine. The absorption of the liposomes by perfused rat liver has been studied as a function of blood level (0-7% haematocrit). It has been found that the rate constant for uptake of liposomes (perfusion constant, kp) is markedly reduced by addition of blood to the perfusate particularly in the haematocrit range 0-3%. The perfusion constant is dependent on the liposome composition and decreases with incorporation of PI and increases with incorporation of stearylamine into DPPC liposomes, but is independent of the initial size of the liposomes in the range of weight-average diameter from 40-400 nm. The possible effects of blood components on the liposomes and their subsequent absorption by the liver are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

The effect of surface-bound protein on the permeability of proteoliposomes.

Proteoliposomes have been prepared from mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol by sonication (SUV) and reverse phase evaporation (REV) and conjugated with succinyl concanavalin A (sConA). The proteoliposomes were characterised in terms of size and composition and covered a range of size (weight-average diameter) from approx. 80 to 300 nm and surface-bound sConA (weight-average number of protein molecules per liposome) from approx. 200 to 1800. The permeabilities of the proteoliposomes to encapsulated D-glucose have been measured and found to increase linearly with protein conjugation. The D-glucose permeability also increases with temperature and passes through a maximum in the region of the gel to liquid-crystalline phase transition temperature. Conjugation has no effect on the chain-melting temperature but slightly decreases the enthalpy of the transition consistent with the withdrawal of some phospholipid participation in chain-melting. The D-glucose permeabilities and thermotropic properties of the proteoliposomes are discussed in terms of the dislocation of the bilayer by the possible off-axis motion of the lipid which anchors the protein to the liposomal surface.

Concanavalin A↗
PubMed1990

A simple theoretical treatment of a competitive enzyme-linked immunosorbent assay (ELISA) and its application to the detection of human blood group antigens.

A theory has been developed to explain the behaviour of a competitive enzyme-linked immunosorbent assay (ELISA) in which an immobilized antigen competes with a liquid-phase antigen for a limiting amount of antibody. The binding of antibody to antigen on a solid surface (microtitre well) is described in terms of Langmuirian adsorption with a binding constant kappa. Two equations are presented to describe the behaviour of the ELISA signal as a function of competing antigen concentration; an exact equation and an approximate equation which can be used when the surface coverage of the immobilized antigen is not known. It is shown how curves of ELISA signal vs. competing antigen concentration depend on K/kappa [antibody]. The theory has been tested using several immobilized blood group A antigens competing with ovarian cyst fluid A substance and found to adequately describe these competitive ELISAs which have a detection limit of approximately 1 ng of blood group antigen.

Binding, Competitive↗
PubMed1990

The role of insulin receptor sulphydryl groups in insulin binding and cellular response in rat adipocytes.

Phenylarsine oxide (PAO), an agent which reacts with vicinal sulphydryl groups and dithiothreitol (DTT), a disulphide reducing agent, inhibited insulin binding to intact adipocytes with half maximal inhibition occurring at 28 microM and 340 microM, respectively. Pretreatment of adipocytes with DTT (2mM) prevented insulin stimulation of glucose uptake by approximately 50%. The marked inhibition of insulin binding to adipocytes by PAO and DTT is consistent with the involvement of the receptor cysteine-rich region of hormone binding. Furthermore, DTT inhibition of insulin binding suggests that the integrity of disulphide bridges is critical for insulin binding. The inhibitory effect of DTT and PAO on insulin binding were not additive, instead addition of DTT to PAO-treated adipocytes effected 15% reversal of binding inhibition. The marked inhibition of insulin binding by addition of low concentrations of DTT (0.2-2.0mM) to intact adipocytes is in contrast to the previously reported biphasic response for the effect of DTT on insulin binding to isolated plasma membranes from rat adipocytes (Schweitzer et al. Proc. Natl. Acad. Sci. U.S.A. 77, 4692-4696, 1980). Scatchard plots for 125I-iodoinsulin binding to adipocytes in the basal state were linear. In contrast, Scatchard analysis of insulin binding to plasma membranes prepared from both basal and insulin-stimulated adipocytes yielded severely curvilinear plots. The data suggests that (i) fundamental differences exist between the receptor state in intact cells and isolated plasma membranes and (ii) that a disulphide-rich region within the insulin receptor, other than the previously reported class I and class II disulphide bridges, is critical for insulin binding and cellular response.

Adipose Tissue↗
PubMed1990

Phenylarsine oxide and the mechanism of insulin-stimulated sugar transport.

The actions of phenylarsine oxide (PAO) on hormone receptors and transport processes are reviewed with particular reference to the mechanism of insulin-stimulated sugar transport. It is suggested that as well as reaction with vicinal -SH groups, vicinal -SH/-OH and -SH/-CO2H groups should also be considered as potential reaction sites for PAO. The relatively high levels of these vicinal combinations of groups in many hormone receptors makes them particularly susceptible to reaction with PAO. In the case of insulin-stimulated sugar transport PAO does not inhibit insulin binding to its receptor at low concentrations but may react directly with the glucose transporters in some cells. A hypothesis is proposed suggesting that PAO may react specifically with one transporter isoform (GLUT-4) which is found almost exclusively in rat adipocytes, skeletal muscle and heart tissue (i.e. insulin responsive tissue) whereas in insulin unresponsive cells such as erythrocytes the GLUT-1 isoform is the predominant transporter which is not inhibited by PAO.

Adipose Tissue↗
PubMed1989

Insulin processing and signal transduction in rat adipocytes.

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.

Adipose Tissue↗
PubMed1989

SED88: a Pascal program for the analysis of sedimentation equilibrium data.

Analytical ultracentrifugation is commonly used for the determination of molecular weights (sedimentation equilibrium) and sedimentation coefficients (sedimentation rate) of biological macromolecules in solution. A Turbo Pascal program for the analysis of sedimentation equilibrium centrifugation data produced by absorbance optical systems is described. The user may enter data from a scan of absorbance versus distance from the centre of rotation, via a graphics tablet (or ASCII file). This is subsequently manipulated to yield an apparent weight average molecular weight for the given sample. Plots of ln (absorbance) versus (radius2) may also be produced. The method described uses readily available computational equipment requiring only a graphics tablet in addition to an IBM PC compatible computer. This technique and the software developed have been used to investigate the molecular weight range of two International Humic Substances Society (IHSS) reference samples from the Suwannee River.

Absorption↗