CCK-58: A novel reagent for studying the regulation of cholecystokinin bioactivity

CCK-58 has been shown to be the major circulating form of the hormone in the dog and human. To date, there have been no reports on its biological activity in vivo. We report here that CCK-8 and CCK-58 were equipotent in decreasing gastric motor function after bolus doses and in stimulating protein secretion after continuous infusion in urethane-anesthetized rats. The present results are the first on the in vivo activity of CCK-58, and indicate that because CCK-58 is equipotent to CCK-8, and because it is a major released and circulating form, it may be considered as a major contributor to the expression of cholecystokinin bioactivity.     

A method for molecular phylogeny construction by direct use of nucleotide sequence data

Summary A method for molecular phylogeny construction is newly developed. The method, called the stepwise ancestral sequence method, estimates molecular phylogenetic trees and ancestral sequences simultaneously on the basis of parsimony and sequence homology. For simplicity the emphasis is placed more on parsiomony than on sequence homology in the present study, though both are certainly important. Because parsimony alone will sometimes generate plural candidate trees, the method retains not one but five candidates from which one can then single out the final tree taking other criteria into account.The properties and performance of the method are then examined by simulating an evolving gene along a model phylogenetic tree. The estimated trees are found to lie in a narrow range of the parsimony criteria used in the present study. Thus, other criteria such as biological evidence and likelihood are necessary to single out the correct tree among them, with biological evidence taking precedence over any other criterion. The computer simulation also reveals that the method satisfactorily estimates both tree topology and ancestral sequences, at least for the evolutionary model used in the present study.     

An automated algorithm for sequence confirmation of chemically modified oligonucleotides by tandem mass spectrometry

We have developed a tandem mass spectrometry (MS/MS) data analysis program for confirmation of sequence of chemically modified oligonucleotides. The method is based on the analysis of deconvoluted MS/MS data for fragment ions from three charge states and comparison of these data against a set of computer-generated masses from expected fragmentation patterns. The algorithm compares the experimental masses not only against the fragment set predicted for the expected sequence but also against a wider test set covering all next-neighbor position switches of the original sequence and all pairwise swaps of nucleosides, which in synthesis would result in molecules with masses within a preset mass tolerance. The algorithm is capable of identifying incorrect sequences that would not be distinguished by identity testing with electrospray ionization mass spectrometry. The method has been tested with permutations of the two 21-mer single strands of a chemically modified short interfering RNA containing 2′-O-methyl and phosphorothioate linkages. For both strands, challenge sequences were synthesized and tested with the premise that they were the original sequences. The algorithm correctly reported the locations of next-neighbor position switches and nucleoside swaps. The results confirm the approach as useful for MS/MS-based identity test methods for synthetic oligonucleotides.     

A method for N-terminal de novo sequence analysis of proteins by matrix-assisted laser desorption/ionization mass spectrometry

A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the N^α-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing.