Biomedical subjects

Terry D Lee

Publications and source records attributed to Terry D Lee.

7 recordsLinked to original sources

PubMed2003

Synthesis of biologically active canine CCK-58.

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity.

Amino Acids↗

PubMed2003

Rat progastrin processing yields peptides with altered potency at the CCK-B receptor.

Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity.

Amino Acid Sequence↗

PubMed2003

Monitoring cyclosporin in blood: between-assay differences at trough and 2 hours post-dose (C2).

With the introduction of a cyclosporin monitoring strategy based on the use of a sample collected 2 hours after dosing (C2) rather than the predose sample (C0), there was concern that the differences in blood cyclosporin results from the various assay systems would result in assay-specific target ranges for C2 monitoring. In addition, it was not known if the different proportion of cyclosporin metabolites in the blood 2 hours after dosing compared with that seen in predose samples would alter the relationship between the various assay methodologies. The aim of this study was to address these issues using blood samples from patients who had undergone kidney and liver transplantation. To do this, paired samples were collected predose and 2 hours after cyclosporin dosing at various periods following transplantation in kidney (88 paired samples) and liver (165 paired samples) transplant recipients. Cyclosporin was measured in these samples using five different immunoassays (radioimmunoassay, two fluorescent polarization immunoassays, and two homogeneous immunoassays) and high-performance liquid chromatography-mass spectrometry. The results of the study showed that when using these immunoassays to measure blood cyclosporin concentrations at C0, the cross-reactivity of the antibodies in the different immunoassay kits resulted in target therapeutic ranges that would need to vary between assays to maintain parity. However, when the same assays were used to measure the blood cyclosporin concentration at C2, the results were congruent, and assay-specific target therapeutic ranges should not be necessary. Thus, when adopting a C2 monitoring strategy, it is possible to use target therapeutic ranges that are independent of the assay system used.

Chromatography, High Pressure Liquid↗

PubMed2002

DNA affinity capture and protein profiling by SELDI-TOF mass spectrometry: effect of DNA methylation.

We report here that surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, as performed on a Ciphergen Biosystems ProteinChip System, can be used in conjunction with DNA affinity capture (DACA) to study specific DNA-protein binding. Using DNA molecules bound to a surface, sequence-specific interactions can be detected as demonstrated by a mutation affecting the binding profile for TBP, a transcription factor. Also, a comparison between methylated and unmethylated promoter-containing DNA fragments shows numerous binding profile differences over a mass range extending to >60 kDa. The binding of several proteins is inhibited by methylation of the DNA.

Base Sequence↗

PubMed2002

Qscore: an algorithm for evaluating SEQUEST database search results.

A scoring procedure is described for measuring the quality of the results for protein identifications obtained from spectral matching of MS/MS data using the Sequest database search program. The scoring system is essentially probabilistic and operates by estimating the probability that a protein identification has come about by chance. The probability is based on the number of identified peptides from the protein, the total number of identified peptides, and the fraction of distinct tryptic peptides from the database that are present in the identified protein. The score is not strictly a probability, as it also incorporates information about the quality of the individual peptide matches. The result of using Qscore on a large test set of data was similar to that achieved using approaches that validate individual spectral matches, with only a narrow overlap in scores between identified proteins and false positive matches. In direct comparison with a published method of evaluating Sequest results, Qscore was able to identify an equivalent number of proteins without any identifiable false positive assignments. Qscore greatly reduces the number of Sequest protein identifications that have to be validated manually.

Algorithms↗

PubMed2002

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Protein Biosynthesis↗

PubMed2002

Differences in receptor binding and stability to enzymatic digestion between CCK-8 and CCK-58.

INTRODUCTION AND AIMS: It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus. METHODOLOGY: Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11. RESULTS: CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not. CONCLUSIONS: The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK-8 and CCK-58 influence receptor binding and stability to enzymatic digestion.

Amino Acid Sequence↗