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Biomedical subjects

R E Offord

Publications and source records attributed to R E Offord.

At least 91 records · Page 5Linked to original sources
PubMed1977

A second activation peptide from bovine cationic trypsinogen.

1. Although only one activation peptide of bovine cationic trypsinogen has been reported previously, the peptide fraction obtained from activation mixtures shows several bands on paper electrophoresis at pH 6.5. 2. The major band was the peptide previously described. The band second in intensity of staining with ninhydrin (10-20% of that of the main band, as judged by eye) had an electrophoretic mobility consistent with its being related to the main peptide. It appeared on activation both of bulk commercial samples of trypsinogen and, as the Appendix shows, of samples prepared from pancreases obtained at the local abattoir. 3. The second peptide proved to be Phe-Pro-Val-Asp-Asp-Asp-Asp-Lys, and we conclude that it is another activation peptide. We discuss briefly the genetic and phylogenetic implications of our findings.

Amino Acid Sequence↗
PubMed1977

Skin test for coeliac disease using a subfraction of gluten.

Gluten and various of its fractions and subfractions have been used for intradermal testing in 10 patients with coeliac disease and in 20 healthy control subjects. All the coeliac patients gave positive Arthus-type reactions (type III) to the subfractions of gluten, whereas all the control subjects were negative. The subfraction B2 evoked strong reactions in almost all the coeliac subjects. If tests with larger series confirm the present results, skin testing with B2 may prove to be a useful screening test for coeliac disease. As B2 has already been found to stimulate the lymphocytes of coeliac patients, the present findings are also of interest in relation to the pathogenesis of coeliac disease.

Adolescent↗
PubMed1977

A functioning complex between tryptic fragments of cytochrome c. A route to the production of semisynthetic analogues.

We report the discovery of a functioning non-covalent complex between two peptides obtained from a limited tryptic digest of horse heart cytochrome c. We have used the phenomenon to produce three modified versions of the complex. We have replaced lysine-39 semisynthetically with ornithine in the first analogue, with p-fluorophenyl-alanine in the second, and removed it entirely in the third.

Animals↗
PubMed1977

A novel semisynthetic route to insulin analogues modified at the N-terminus of the A-chain.

We report a novel route for the semi-synthetic production of insulin analogues modified at the amino-terminus of the A-chain. The route proceeds from insulin via [Nalpha-Boc-GlyA1]-insulin to [Nalpha-(Z-methionyl)-PheB1, Nepsilon-(Z-methionyl)-LysB29]des-GlyA1-insulin. We have replaced residue A1 with glycine, to give a fully crystalline resynthesized insulin, and with D-lysine.

Amino Acids↗
PubMed1976

The preparation of protected fragments of lysozyme for semisynthesis.

This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain...

Acylation↗
PubMed1976

The semisynthesis of portions of hen's-egg lysozyme by fragment condensation.

1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion.

Amino Acid Sequence↗
PubMed1976

The degradation of semisynthetic tritiated insulin by perfused mouse livers.

Semisynthetic [3H]insulin was stored under various conditions for up to 180 days and the stability of the insulin under these conditions was assessed. A sample that had been stored for 180 days was repurified and shown to be degraded at the same rate as native insulin by perfused mouse livers, even at low physiological concentrations. After perfusion, intact insulin could be separated from degradation products, and the radioactivity associated with the insulin fraction could be used to determine the percentage degradation. The initial rate of degradation of insulin was a linear function of concentration over the range 360pM-1.9nM.

Animals↗
PubMed1976

The separation and sequencing of permethylated peptides by mass spectrometry directly coupled to gas-liquid chromatography.

The use of g.l.c. coupled to mass spectrometry to separate and sequence permethylated acetyl- and trifluoroacetyl-peptides in a single operation is described. Both electron impact and chemical ionization were used to induce fragmentation, and the latter was found to be more sensitive. Chromatographic retention data are presented which suggest that peptide derivatives of molecular weight of at least 750 are accessible to the technique. The application of our methods to the determination of the primary sequence of proteins is discussed.

Chromatography, Gas↗
PubMed1976

Stimulation of lymphocytes from patients with coeliac disease by a subfraction of gluten.

Peripheral-blood lymphocytes from patients with coeliac disease showed a marked increase in D.N.A. synthesis after incubation in vitro with a subfraction of gluten. Lymphocytes from control patients showed no increased D.N.A. synthesis under the same conditions. The possible use of this specific lymphocyte-stimulation assay as a screening test for coeliac disease is discussed.

Adolescent↗
PubMed1975

The preparation of a semisynthetic tritiated insulin with a specific radioactivity of up to 20 Curies per millimole.

1. This paper describes the semisynthesis of [3H]insulin of high specific radioactivity which is indistinguishable from native insulin in its hypoglycaemic activity towards mice. 2. The maximum specific radioactivity so far obtained is 20 Ci/mmol. At this value the recommended scale of preparation is between 1 mCi (30 mug of product) and 10mCi (300 mug of product). Reaction volumes are then of the order of 10 mul. 3. Special problems arise when the techniques of peptide synthesis are applied to the production of such small quantities of material. We describe these problems and discuss the means of overcoming them.

Animals↗
PubMed1974

Studies on the subunit structure and amino acid sequence of trisoe phosphate isomerase from chicken breast muscle.

1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH(4))(2)SO(4) fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E(0.1%) (280) is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

Amino Acid Sequence↗
PubMed1974

Identifying toxic fractions of wheat gluten and their effect on the jejunal mucosa in coeliac disease.

THE TOXICITY OF THREE FRACTIONS (A, B, AND C) OBTAINED BY ULTRAFILTRATION OF A PEPTIC: tryptic digest of gluten has been assessed by serial feeding experiments in patients with treated coeliac disease. The first fraction (A), which contains amino acids and oligopeptides, produced no damage to the jejunal mucosa. The other two fractions (B and C) both caused mucosal damage. Fraction B, which contains the products of digestion of smaller molecular weight, consists of polypeptides which are concentrated in the region of 8000 molecular weight. It contains no gliadin (molecular weight 50 000) or gluten. Ultrastructural evidence of damage was visible six hours after challenge with fraction B and by 10 hours histological abnormalities were also present. Ultrastructural abnormalities occurred early in the epithelial cells and preceded changes in the basement membrane and capillaries. The disaccharidases showed a pronounced depression in all three subjects by 24 hours. The rapid onset of damage after challenge, coupled with the evidence of recovery as soon as 72 hours later, is more in keeping with a direct action on the surface epithelial cells rather than an immune mechanism.

Amino Acids↗