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J E Shields

Publications and source records attributed to J E Shields.

At least 19 recordsLinked to original sources
PubMed1997

Modifications in the B10 and B26-30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor.

Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40-60% relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5-2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28-29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31-32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin.

Amino Acid Sequence↗
PubMed1997

Bioavailability and bioeffectiveness of subcutaneous human insulin and two of its analogs--LysB28ProB29-human insulin and AspB10LysB28ProB29-human insulin--assessed in a conscious pig model.

In this study, human insulin was compared with two of its analogs--LysB28ProB29-human insulin and AspB10LysB28ProB29-human insulin-with respect to bioavailability and metabolic effectiveness. Absorption from the subcutaneous site was determined using kinetic parameters from the washout curve, following intravenous infusion of insulin or analog. Absorption was found to be more rapid for the two analogs, with 90% absorption by 100 min for the analogs and by 180 min for insulin. Total absorption was 97 +/- 10% for insulin, 99 +/- 7% for LysB28ProB29-human insulin, and 93 +/- 12% for AspB10LysB28ProB29-human insulin. Bioactivity was assessed from the glucose infusion and using tracer-determined metabolic clearance rates (MCRs) and glucose production rates. The fractional glucose requirements (relative to the total amount infused) increased more rapidly for the two analogs than for insulin, with 50% of the glucose infused by 105 min for both analogs vs. 145 min for insulin. The total amount of glucose required was, however, significantly less (19.7 +/- 1.5 mmol/kg) for AspB10LysB28ProB29-human insulin than for either LysB28ProB29-human insulin (25.9 +/- 3.0 mmol/kg) or human insulin (27.8 +/- 2.6 mmol/kg). The glucose requirements were reflected in a lower MCR for AspB10LysB28ProB29-human insulin but equivalent decreases in the rates of glucose production. Thus both analogs demonstrated more rapid rates of absorption, onset, and termination of action, but were not completely bioequivalent, with AspB10LysB28ProB29-human insulin demonstrating a 25% decrease in bioactivity.

Absorption↗
PubMed1994

Preparation of an insulin with improved pharmacokinetics relative to human insulin through consideration of structural homology with insulin-like growth factor I.

The Diabetes Control and Complications Trial has emphasized the need for improved control of blood glucose as a means to diminish long-term complications of diabetes. LysPro-insulin is an analog of human insulin whose design was modeled on structural homology with insulin-like growth factor I. An analysis of the structural conformation of insulin suggested that an inversion of amino acids B28 and B29 in the C-terminus of the B chain could yield an insulin analog with a faster onset of biological action. This insulin analog has proved to be virtually identical to human insulin in action, with one important exception. LysPro-insulin has demonstrated an improved time course of action in control of a mealtime glucose elevation. This offers the opportunity for improved convenience and safety for patients with insulin-dependent diabetes mellitus.

Amino Acid Sequence↗
PubMed1992

pH dependent conformational changes in the T- and R-states of insulin in solution: circular dichroic studies in the pH range of 6 to 10.

Zinc insulin hexamer has been shown to undergo a phenol-induced T6 to R6 conformational transition in solution. Our circular dichroic (CD) studies demonstrate that insulin undergoes pH-dependent conformational changes over the pH range of 6-10 in the T-state and in the R- state. In order to determine which specific amino acid residues may be responsible for these pH-dependent changes, a series of insulin analogs were utilized. In the T-state, the pH dependent CD changes monitored in the far UV region have a pK of 8.2 and appear to be related to the titration of the A1-Gly amino group. Using the near UV CD a second pH-dependent conformational change was detected with a pK of 7.5 in the T-state. 1H N.M.R. studies suggest that B5-His may be responsible for this conformational transition. In the presence of m-cresol (R-state), the pK value was found to be 6.9. During this titration, the increased ellipticity for the R-state is diminishing as pH decreases from pH 8 to 6, and no difference in ellipticity was observed at 255 nm between T- and R-states at pH 6. Therefore, this may be due to the transition from the R back to the T-state.

Circular Dichroism↗
PubMed1992

Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.

Amino Acid Sequence↗
PubMed1992

Altering the association properties of insulin by amino acid replacement.

The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.

Amino Acid Sequence↗
PubMed1989

Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer.

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.

Amino Acid Sequence↗
PubMed1983

Synthetic Fc peptide-mediated regulation of the immune response. I. Characterization of the immunomodulating properties of a synthetic 23-amino acid peptide derived from the sequence of the CH3 domain of human IgG1.

A synthetic 23-amino acid peptide derived from the CH3 domain of human IgG1 was found to be a potent adjuvant as well as a polyclonal activator. The Fc peptide was found to enhance human and murine humoral, and T cell-mediated immune responses. Moreover, in vivo administration of Fc peptide enhanced murine natural killer cell activity. The synthetic Fc peptide was found to be more potent, on a molar basis, than native Fc fragments in inducing polyclonal antibody production and potentiating immune responses.

Adjuvants, Immunologic↗