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Biomedical subjects

P E Thompson

Publications and source records attributed to P E Thompson.

At least 19 recordsLinked to original sources
PubMed2001

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.

Amino Acid Sequence↗
PubMed2001

Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH(6-13): comparison of two i-->i + 4 lactam cyclization procedures.

The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N-terminus of human growth hormone (hGH), namely hGH[6-13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse beta-turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric byproducts associated with the preparation of cyclic hGH[6-14] peptide analogues based on an i-->(i + 4)Lys-->Glu or Glu-->Lys cyclization strategy.

Amino Acid Sequence↗
PubMed1999

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.

Amino Acid Sequence↗
PubMed1998

Comparison of the binding of alpha-helical and beta-sheet peptides to a hydrophobic surface.

The induction and stabilisation of secondary structure for a series of amphipathic alpha-helical and beta-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from beta-sheet to alpha-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85 degrees C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.

Amino Acid Sequence↗
PubMed1997

Conformational stability of a type II' beta-turn motif in human growth hormone [6-13] peptide analogues at hydrophobic surfaces.

The interactive properties of several peptides related to human growth hormone (hGH) [6-13] containing a type II' beta-turn motif have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC). Various chromatographic parameters related to the hydrophobic interactive surface area and binding affinity were measured over the range of temperatures between 5 and 85 degrees C. Variations in these parameters were consistent with significant differences in the relative stability of the type II' beta-turn structures of these peptidomimetics. The effect of changes in peptide conformation were also investigated through the analysis of band-broadening behaviour during the chromatographic process. Significant variations in bandwidth observed at discrete temperatures were related to the rate of interconversion between the type II' beta-turn and more extended conformers. These investigations further document the potential of RP-HPLC for monitoring subtle changes in peptide secondary structure at hydrophobic interfaces.

Chromatography, High Pressure Liquid↗
PubMed1997

Nurse managed prenatal programs affect outcomes for corporations.

Faced with higher medical costs and increased insurance premiums, corporations are focusing on health promotion and wellness. With increasing numbers of women in the workforce, corporations have identified the need for prenatal programs. By developing, initiating, and evaluating outcome-based prenatal programs nurses can target the health care needs of this select population. One such program documented several outcomes including improved employee health and an 86% reduction in maternal/newborn costs.

Cost-Benefit Analysis↗
PubMed1996

Conformational analysis of human growth hormone [6-13] peptide analogues.

The conformational analysis of a series of ten hGH[6-13] peptide analogues is reported. As part of our earlier studies, the alpha-aminosuccinimide modified fragment Asu11-hGH[6-13] has previously been identified as a potentiator of insulin activity in intravenous insulin tolerance tests, and various analogues have been subsequently designed, synthesised and employed to acquire structure-activity data. These studies have lead to the conclusion that the conformational characteristics at the C-terminus of each of the active peptide analogues is important to the biological activity. In the present investigation, molecular dynamics and simulated annealing techniques have been used to examine the accessible conformational states of the C-terminal region of ten different hGH[6-13] peptide analogues. Of these six are active peptide analogues while the other four show no biological activity. Examination of the conformer groups identified using this molecular dynamics approach showed a common conformational motif for each of the active peptides.

Growth Hormone↗
PubMed1995

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples.

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.

Adsorption↗
PubMed1995

Synthesis of peptide amides using Fmoc-based solid-phase procedures on 4-methylbenzhydrylamine resins.

A two-step low-high protocol for the efficient synthesis of peptide amides is described. The protocol exploits the efficiency of Reagent K for side-chain deprotection with the capability of the hard acid trifluoromethane-sulfonic acid (TFSMA) for cleavage of the peptide from the benzhydrylamine resin. This procedure has proven to be an effective method for the synthesis of peptide amides. The formation of alpha-aminosuccinimide (Asu) derivatives were observed with aspartyl-containing peptides as a minor side reaction product of this procedure, but this Asp-->Asu rearrangement could be successfully suppressed by employing low temperature conditions. The N- to O-acyl rearrangement of threonine and/or serine residues also only occurred to a minor extent under these synthetic conditions.

Amides↗
PubMed1992

Conformational studies on (+)-anatoxin-a and derivatives.

Anatoxin-a (AnTX) is a highly potent agonist acting at the nicotinic acetylcholine receptor (nAChR) and represents a valuable tool in the study of this receptor. Molecular mechanics, semi-empirical and ab initio molecular orbital energy minimization procedures were conducted to investigate the conformation of AnTX. For each minimization procedure, the s-trans enone isomer of protonated AnTX was the energetically favoured conformer due to intramolecular electrostatic interactions. Our studies are discussed in the light of previous experimental observations and conformational studies, in addition to their importance in the development of future pharmacophore models for nAChR agonist binding.

Bacterial Toxins↗
PubMed1991

Antibodies to the ganglioside GD1b in a patient with motor neuron disease and thyroid adenoma.

Patients with motor neuron disease with thyroid disorders have been described, although the relationship between the two conditions is unclear. We treated a patient with amyotrophic lateral sclerosis who also had a follicular adenoma of the thyroid gland. Because thyroid gland plasma membranes contain high concentrations of complex gangliosides, such as GD1b, and some patients with motor neuron disease have IgM antibodies to GD1b, we decided to assay serum from this patient for the presence of antiganglioside antibodies. IgM antibodies to GD1b were detectable at serum dilutions of 1:500 and 1:1000 by enzyme-linked immunosorbent assay. While these titers are less than those usually described in patients with plasma cell dyscrasia, they are well in excess of normal values. Antibody to GM1 was also detectable at a lower (1:100) dilution. We do not know the importance of the anti-GD1b antibodies in this patient, but it is possible that antibodies to GD1b are involved in this and other cases of motor neuron disease associated with thyroid disease.

Adenoma↗
PubMed1980

Immunoreactive helix-destabilizing protein localized in transcriptionally active regions of Drosophila polytene chromosomes.

A highly purified helix-destabilizing protein (HDP) obtained from rat liver has been used to elicit specific, high-titer anti-HDP sera in rabbits. These antisera show immunological crossreaction with single-stranded DNA binding proteins from several very diverse eukaryotic sources, including Drosophila embryos. The use of such antisera in the labeling of Drosophila salivary gland chromosomes by indirect immunofluorescence shows concentrations of immunoreactive HDP in many regions, but especially in chromosome puffs. There is a striking localization of HDP in heat shock puffs known to be sites of new transcription. The pattern of HDP distribution seems to implicate a transcriptional role, with some specificities independent of puffing itself.

Animals↗
PubMed1976

Two-nucleotide codon change in a hemoglobin polymorphism of the Celebes black ape (Macaca nigra).

A hemoglobin polymorphism involving variant beta-chains was demonstrated in the Celebes black ape, Macaca nigra. Fingerprinting and amino acid analysis of the tryptic peptides from the two chain types have shown that they differ by a single amino acid substitution, between lysine and aspartic acid, which requires a two-nucleotide change in the corresponding codon. Another substitution in the same codon is found as a species between the black ape and that of other macaques.

Amino Acids↗