We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.

Amino Acid Sequence↗

PubMedDate not supplied

Biotransformation of the antiemetic 5-HT3 antagonist tropisetron in liver and kidney slices of human, rat and dog with a comparison to in vivo.

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.

Administration, Oral↗

PubMedDate not supplied

Purification and characterization of an exon 2-deleted human beta-tropomyosin constructed by the polymerase chain reaction.

We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.

Base Sequence↗

PubMedDate not supplied

Improvement of porphyrin cellular delivery and activity by conjugation to a carrier peptide.

The chemical nuclease metalloporphyrin (manganese(III) porphyrin) can cleave DNA irreversibly and can thus constitute a potential antitumor drug. However, these molecules show low permeability to cell surface membranes. We report here the conjugation of an amphipathic carrier peptide to improve considerably its cellular delivery. The metalloporphyrin-peptide conjugate can be internalized by cells within only 5 min of incubation with a yield as high as 80%. Furthermore, the metalloporphyrin-peptide conjugate is able to cleave in vitro high or low molecular weight DNA to the same extend as metalloporphyrin alone without affecting the sequence-specific cleaving activity of the porphyrin. The conjugate is 100-fold more efficient at inducing tumor cells death than the free metalloporphyrin via a mechanism involving genomic DNA cleavage. The results are promising for further therapeutic applications with antitumor drugs such as metalloporphyrin, and also with other existing drugs by using a carrier peptide system in order to improve the cellular uptake of such molecules.

Amino Acid Sequence↗