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Biomedical subjects

M D Barratt

Publications and source records attributed to M D Barratt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Induction of photoallergy in guinea-pigs by injection of photoallergen-protein conjugates.

Photoconjugates were prepared by ultraviolet irradiation of guinea-pig albumin (GPA) with the photoallergens tetrachlorosalicylanilide (T4CS) and fentichlor. Injections of T4CS-GPA induced photoallergy to T4CS in 11 of 12 guinea-pigs whereas injections of fentichlor-GPA induced photoallergy in 5 of 12 guinea-pigs. Thus the fentichlor-GPA photoconjugate, which contained a molar ratio of hapten to protein 3 times higher than the corresponding T4CS conjugate, produced a significantly lower response. The results demonstrate the importance of protein conjugate formation in the induction of photoallergy, i.e. the role of carrier protein in contact sensitivity. The high potency of the T4CS-GPA photoconjugate in inducing photoallergy suggests that albumin may have a special role as a carrier protein in T4CS photoallergy.

Albumins↗
PubMed1985

Photochemical binding of photoallergens to human serum albumin: a simple in vitro method for screening potential photoallergens.

A simple procedure employing UV spectroscopy is described for testing the ability of chemicals to form covalent conjugates with proteins after irradiation with the appropriate wavelength of light. A range of known photoallergens of widely differing structure has been tested using this procedure; results of these experiments, together with evidence from the scientific literature, provide a correlation between compounds known to be photoallergens and their ability to form covalent conjugates with proteins on irradiation with the appropriate wavelength of light. The method is proposed as an in vitro screening procedure for potential photoallergens.

Allergens↗
PubMed1984

Binding of a spin label analogue of compound 48/80 to rat peritoneal mast cells: correlation of binding properties with surface topography.

A spin label analogue of compound 48/80 has been synthesized and its binding to purified rat peritoneal mast cells has been studied by electron spin resonance spectroscopy. The spin label analogue (SL-48/80) had almost identical biological activity to unlabeled compound 48/80. SL-48/80 was used to estimate the number of binding sites per cell on normal mast cells (7.25 X 10(10)), on mast cells deactivated by sodium azide and 2-deoxyglucose or by heating to 46 degrees for 30 min (1 X 10(10)) and cells from animals actively-sensitized to ovalbumin (5.2 X 10(10)). SL-48/80 was also shown to bind to isolated mast cell granules. Differences in the binding properties of mast cells after the different treatments are related to their surface topography as seen by scanning electron microscopy, and the contribution of the granules to the number of binding sites is discussed.

Animals↗
PubMed1984

Photochemical reactions of fentichlor with soluble proteins.

The photochemical reactions of the photoallergen fentichlor with soluble proteins have been studied. [35S]Fentichlor was shown to bind covalently to human serum albumin (HSA) when irradiated with UV light (313 nm). HSA had the ability to bind at least eight molecules of fentichlor per molecule of protein. Fractionation of fentichlor-HSA photoadducts after (a) treatment with cyanogen bromide and (b) reduction, carboxymethylation and digestion with trypsin showed that the bound fentichlor was distributed fairly evenly throughout the sequence of the HSA molecule. Fentichlor was also shown to form photoadducts with human gamma-globulin and with bovine insulin. Its binding to insulin was restricted to the B chain of the molecule. Fundamental differences between the photochemical reactions of the photo-allergens fentichlor and tetrachlorosalicylanilide (T4CS) with soluble proteins are discussed. The reactions of fentichlor with soluble proteins are not restricted to specific binding sites (unlike T4CS). Fentichlor has the potential to react photochemically with a wide range of proteins in the epidermis and dermis, to form antigens.

Allergens↗
PubMed1984

Binding of a spin-labelled photoallergen to human serum albumin.

The binding site for 3,3',4',5-tetrachlorosalicylanilide (T4CS), a potent photoallergen, on human serum albumin (HSA) was studied by electron spin resonance spectroscopy using a spin-labelled analogue 3,5-dichlorosalicylamido-4-(2,2,6,6-tetramethylpiperidine 1-oxyl) (DCS-TEMPO) of T4CS in the absence of ultraviolet irradiation. DCS-TEMPO bound non-covalently (K = 5.8 X 10(6) M-1) to one major binding site on HSA. This binding site could be blocked by the photochemical binding of T4CS to the protein. Limited tryptic digestion of HSA or chemical modification of its single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide was found to reduce the binding constant of the T4CS/DCS-TEMPO-binding site. These observations are in good agreement with earlier conclusions on the nature of the T4CS-binding site and suggest a location for this site close to the single tryptophan residue of the HSA molecule.

Allergens↗
PubMed1983

The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus.

The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.

Electron Spin Resonance Spectroscopy↗
PubMed1982

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes.

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.

Allergens↗
PubMed1979

The interaction of 3,3',4',5-tetrachlorosalicylanilide with phosphatidylcholine bilayers.

1. The interaction of the germicide 3,3',4',5-tetrachlorosalicylanilide (T4CS) with vesicles and dispersions of egg phosphatidylcholine has been studied by gel permeation chromatography, electron microscopy, electron spin resonance spin labelling and ion permeability measurements. 2. Incorporation of T4CS into vesicles of egg phosphatidylcholine gives rise to a large increase in the permeability rate of the paramagnetic cation N,N-dimethyl-N-(1'-oxyl-2',2',6',6'-tetramethyl-4'-piperidyl)-2-hydroxyethylammonium chloride through the lipid bilayer but has no significant effect on the vesicle sizes as measured by gel permeation chromatography or electron microscopy. 3. ESR studies using a spin-labelled fatty acid have demonstrated the presence of two different environments for the spin label when T4CS is incorporated into phosphatidylcholine bilayers. These two environments are identified as (a) highly ordered areas of the bilayer, rich in T4CS and (b) areas with very similar ordering to that in pure egg phosphatidylcholine. 4. The effectiveness of very low concentrations of the germicide in increasing vesicle permeability is explained in terms of its clustering to give rigid patches, rich in T4CS, rather than being evenly distributed throughout the bilayer. It is proposed that the increased ion permeability arises from leakage at the interfaces between the rigid and flexible regions of the lipid bilayer. 5. Comparisons between the effective levels of T4CS in phosphatidylcholine vesicles and its minimum inhibitory concentration with a Gram-positive bacterium confirm the validity of phospholipid vesicles as a model for studies of germicidal activity.

Chromatography, Gel↗
PubMed1976

Interaction of apoprotein from porcine high-density lipoprotein with dimyristoly lecithin. 2. Nature of lipid-protein interaction.

The detailed molecular structure of the complex formed by the apoprotein from porcine high density lipoprotein and dimyristoly phosphatidylcholine (lecithin) has been investigated by a range of physical techniques. The complex, an oblate ellipsoid with major axis 11.0 nm and minor axis 5.5 nm (see the accompanying paper), is comprised of a section of lecithin bilayer with apoprotein at the surface. The main site of interaction between protein and lipid is in the lipid glycerophosphorylcholine group region; as with native high density lipoprotein the surface of the particle consists of a mosaic of lecithin polar groups and protein. The formation of this mosaic reduces the cooperativity of the lecithin chain motions and changes the curvature of the lipid-water interface, as compared to a bilayer. Otherwise, there are no major changes in lecithin motions indicating that no strong binding of lipid to protein occurs. The interaction involves the intercalation of amphipathic, 60% alpha-helical, apoprotein molecules among the lecithin molecules so that the protein residues at the lipid-water interface. The apoprotein has a high affinity for the lipid-water interface but specific lipid-protein interactions are not involved.

Amino Acid Sequence↗
PubMed1976

A study of Folch-Pi apoprotein. II. Relation between polymerization state and conformation.

A comparison of the conformation of Folch-Pi apoprotein in organic solvent and in aqueous solutions has been made by ESR, infrared and circular dichroism spectroscopy studies. Electrophoresis and ultracentrifugation have been carried out in order to correlate molecular weight and charge of the molecule with its conformation. It appears that the protein is monomeric in organic solution. In water, only one component is present but the molecules behave as a polydisperse system of associating molecules. Hydrophobic interacitons seem to be important for this polymerisation which does not appear to be accompanied by the formation of beta-structure. After the transfer of the protein from organic solution to water, the ESR spectra of the protein labelled on the free SH groups show an heterogeneity in the motional environment of the label which permits to assume that different areas of association exist in the polymeric molecule.

Apoproteins↗
PubMed1975

Differences in the interaction of inorganic and organic (hydrophobic) cations with phosphatidylserine membranes.

The interaction of phosphatidylserine dispersions with "hydrophobic", organic cations (acetylcholine, tetraethylammonium ion) is compared with that of simple inorganic cations (Na+, Ca2+); differences in the hydration properties of the two classes of ions exist in the bulk phase as evident from spin-lattice relaxation time T1 measurements. It is shown that the reaction products (cation-phospholipid) differ markedly in their physicochemical behaviour. With increasing concentration both classes of ions reduce the zota-potential of phosphatidylserine surfaces, the monovalent inorganic cations being only slightly more effective than the hydrophobic cations. Inorganic cations cause precipitation of the lipid once the surface charge of the bilayer is reduced to a certain threshold value. This is not the case with the organic cations. The difference is probably associated with the different hydration properties of the resulting complexes. Thus binding of Ca2+ causes displacement of water of hydration and formation of an anhydrous, hydrophobic calcium-phosphatidylserine complex which is insoluble in water, whereas the product of binding of the organic cations is hydrated, hydrophilic and water soluble. The above findings are consistent with NMR results which show that the phosphodiester group is involved in the binding of both classes of cations as well as being the site of the primary hydration shell. Besides affecting interbilayer membrane interactions such as those involved in cell adhesion and membrane fusion, the binding of both classes of cation can affect the molecular packing within a bilayer.

Acetylcholine↗