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Biomedical subjects

R E Offord

Publications and source records attributed to R E Offord.

At least 73 records · Page 4Linked to original sources
PubMed1983

Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis.

Reaction conditions are described that permit the enzyme-assisted semi-synthetic replacement of residue B30 of pig insulin (or of analogue) to proceed in very high yield in 2 h or less. Immobilized trypsin may be used as catalyst, and excess amino acid ester may be recycled after a simple extraction. Alanine-B30 may be replaced by a variety of nucleophiles, including threonine O-t-butyl ether t-butyl ester, in which case the yield of crude product is about 99%. De-protection of the B30 threonyl ester analogue of insulin thus formed then affords human insulin in an overall yield of about 92%, based on pig starting material. The product has full biological potency, as determined by depression of blood glucose concentration in rats, and showed the expected behaviour on radioimmunoassay.

Animals↗
PubMed1982

The proteinase-catalysed synthesis of peptide hydrazides.

1. We report the trypsin-catalysed conversion, in high yield, of peptides to peptide hydrazides, t-butyloxycarbonylhydrazides and phenylhydrazides. The substitution is at the alpha-carboxy group. 2. We discuss the relative merits of carrying out the conversion either simultaneously with tryptic cleavage of the parent protein or after such cleavage. 3. We report analogous results with chymotrypsin, elastase and subtilisin. 4. We propose the use of such products in protein semi-synthesis and in the preparation of specific proteinase inhibitors.

Catalysis↗
PubMed1981

Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.

Acetylation↗
PubMed1981

The role of the arginine-B22 residue in insulin action.

We describe the modification of the side chain of the arginine-B22 residue of insulin by the N8N9-(1, 2-dihydroxycyclohex-1,2-ylene) group and by the adipoyl group. These are the first insulin derivatives described that contain a modified arginine residue in an otherwise unaltered molecule. When tested for their ability to lower blood sugar concentration, both modified insulins showed a specific activity indistinguishable from that of insulin. In view of the fact that the substituent groups involved are very bulky and in one case of opposite charge to that of the side chain, the retention of biological activity casts doubt on the idea, previously generally accepted, that the arginine-B22 residue is essential to the activity of the hormone.

Animals↗
PubMed1981

The timing of histological damage following a single challenge with gluten in treated coeliac disease.

Two treated subjects and one healthy volunteer have been challenged with a single dose of fraction B of gluten administered intraduodenally. Serial biopsy specimens were obtained by means of a Quinton hydraulically operated biopsy instrument immediately before challenge and repeatedly thereafter. The biopsy specimens were studied by light microscopy, electron microscopy, immunofluorescence, immunoperoxidase staining and disaccharidase assay. The two coeliac subjects showed pronounced mucosal changes which developed a few hours after challenge and which showed the features of an Arthus-type reaction. The healthy volunteer was unaffected. It is concluded that the lesion of coeliac disease is the result of an Arthus-type reaction induced by ingested gluten in susceptible subjects.

Aged↗
PubMed1980

A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.

When a polypeptide containing gamma-carboxyglutamic acid is decarboxylated in 2H2O, residue of (gamma gamma-2H2)glutamic acid are formed. Subsequent proteolytic digestion produces peptides which contain at each site 2H2-substituted and unsubstituted glutamic acid in the same ratio as existed for gramma-carboxy-substitution. The peptides may be identified and this ratio determined by combined gas chromatography--mass spectrometry. We also discuss decarboxylation in 3H2O followed by amino-acid analysis and Edman degradation.

1-Carboxyglutamic Acid↗
PubMed1980

The effect of aprotinin on the absorption of subcutaneously injected regular insulin in normal subjects.

The effect of aprotinin on the absorption of regular insulin was assessed in normal man. Ten units of Actrapid insulin were subcutaneously injected together with 1.4 mg aprotinin (i.e., 0.5 ml of Trasylol) or an equivalent volume of physiologic saline (controls) into the thighs of overnight-fasted normal subjects. Aprotinin caused an increase in the rate of insulin entry into the circulation; the absolute amount of insulin that was detected in the circulation during the course of the experiment was also higher. In addition, the onset of the hypoglycemic action of exogenous insulin was significantly accelerated when insulin was administered together with aprotinin. These data suggest that aprotinin increases the absorption rate of subcutaneously injected insulin from its depot into the circulation, possibly by an inhibition of the local degradation of exogenous insulin at the injection site.

Absorption↗
PubMed1980

Binding and degradation of semisynthetic tritiated insulin by IM-9 cultured human lymphocytes.

Insulin was tritiated by semisynthetic replacement of the amino-terminal phenylalanine of the B chain with tritiated phenylalanine. At 15 degrees C, (3H) insulin bound to high affinity receptors on IM-9 cultured human lymphocytes with an affinity constant of about 3 x 10(9) M-1, The Scatchard plot was curvilinear. At 37 degrees C, maximal binding occurred after about 15 min of incubation. Binding fell thereafter due to degradation of insulin by the extracellular fluid. The major degradation product after 120 min coeluted with insulin from Sephadex G50 and was precipitated by anti-insulin antibody but to a lesser degree than intact insulin. It had little or no biologic activity as assessed by binding to IM-9 lymphocytes. The cell-associated radioactivity was also eluted as a single peak on Sephadex G-50. In contrast to the degradation product, this material retained its ability to bind to insulin receptors. We deduce that this cell-associated material contains the entire A chain, most of the B chain, and is probably native insulin. These data show that insulin bound to IM-9 lymphocytes remains biologically intact.

Antibodies↗
PubMed1979

Inhibition of degradation of insulin by ophthalamic acid and by a bovine pancreatic proteinase inhibitor.

We have previously observed that, on subcutaneous administration, a significant proportion of insulin is degraded at the site of injection. The present paper reports that the degradative activity of slices of rat adipose tissue can be inhibited in vitro by ophthalmic acid, a natural analogue of glutathione, and by bovine pancreatic proteinase inhibitor, whereas it is increased by the addition of reduced glutathione.

Adipose Tissue↗
PubMed1979

The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.

Amino Acid Sequence↗
PubMed1979

Absorption kinetics of subcutaneously injected insulin. Evidence for degradation at the injection site.

The absorption of subcutaneously injected insulin was examined by injecting semisynthetic [3H] insulin in anaesthetized pigs and subsequently analysing the tissue excised from the injection site. Contrary to previously accepted views, a significant proportion of insulin was degraded at the injection site. The disappearance of intact [3H] insulin from the injection site followed a monoexponential function with a half-time of 59 min.

Absorption↗