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↗

PubMedDate not supplied

Side-chain interactions and conformation in alpha-helical poly(gamma-phenacyl L-glutamate). Aggregation in dilute solutions.

The behavior of alpha-helical poly(gamma-phenacyl L-glutamate) (PPLG) has been studied by hydrodynamic, optical rotation, dielectric, and nmr measurements in different solvents. Special attention has been paid to side-chain interactions which depend on the ease of solvation of the phenacyl chromophore. Thus, in hexafluoro-2-propanol the circular dichroism spectrum is very similar to that usually observed for an alpha helix, and hydrogen bonding was shown to occur between the solvent and phenacyl CO group. In other heliocogenic solvents, side chain-side chain interactions occur. In dimethylformamide and pyridine they give rise at low temperature and low concentrations to an aggregation phenomenon whose molecular mechanism implies a molecular weight dependent folding of the molecules. Models for the side-chain conformation are proposed for the polymer in the solid state on the basis of infrared dichroism.

Binding Sites↗

PubMedDate not supplied

alpha and piDL helical states of alternating poly(gamma-benzyl D-L-glutamate) in solution.

As in solid state, strictly alternating poly(gamma-benzyl D-L-glutamate) in solution can adopt two different helical conformations. Besides the alpha helix, a second helical conformation is found at higher temperatures in dioxane and chloroform, the properties of which correspond to that of the piDL4 helix. As the molecules have a finite length a screw sense is favored for both helical forms thus giving rise to optical activity allowing the study of the transconformation by optical rotatory dispersion and circular dichroism besides infrared and dielectric measurements. Thus, as the temperature is raised the equilibria right-left handed alpha helices and alpha-piDL helical forms can be followed. The favored screw senses are determined by the number of interacting side chains for the alpha helix and by the number of hydrogen bonds which are formed in the piDL helical conformation. The side chain-side chain interactions in the alpha helix are experimentally shown to be attractive.

Glutamates↗

PubMedDate not supplied

High-resolution nuclear magnetic resonance studies at 270 MHZ of alternating and random poly(benzyl D,L-glutamates).

The solution conformations of several D,L copoly benzyl glutamates both random and alternating are studied by comparing their NMR spectra in chloroform and also in dioxane and dimethylformamide. The alpha CH chemical shifts characteristic of the alpha helix of strictly alternating DL copolymers in chloroform/0.5% TFA are established (3.65 and 3.82 ppm) and differ from that of the regular alpha helix (3.92 ppm). It is concluded that alternating copolymers prepared by an essentially racemization-free method are completely in the alpha-helical conformation which is characteristic of strictly alternating D-L copolymers, whereas random copolymers are largely regular alpha. The alpha leads to piDL helix/helix transition of an alternating copolymer in dioxane has been monitored and the alpha CH resonance characteristic of the piDL helix is found to be at the unusually low chemical shift of 4.45 ppm.

Glutamates↗

PubMedDate not supplied

Structural properties of double-stranded helical poly (gamma-benzyl-D-L-glutamate) in solution. Comparison with some solution properties of linear gramicidin.

The double-stranded helical conformations of alternating PBD-LG found in the solid state have been studied in infrared, circular dichroism, and NMR techniques in solution in methylene chloride, chloroform dioxane, and collidine. The infrared and CD properties of the solutions, transconformations between single- and double-stranded helices and transconformations within the family of double helices, support the hypothesis that the conformation in solution is the same as that found after evaporation of the solvent, namely the pi pi DL 7.2, pI pI DL 9.0, and pi pi DL 10.8 helices, depending on the solvent. An attempt to identify the conformation of linear gramicidin is made on the basis of the CD spectra and the infrared frequencies conformation relationship established for PBD-LG. However, owing to the great number of different conformations observed for the antibiotic, no firm conclusion can yet be drawn, except for the probable existence of the double antiparallel helical structure.

Benzyl Compounds↗

PubMedDate not supplied

High-resolution NMR studies at 270 MHz of alternating poly(gamma-benzyl D-L-glutamate) in double-stranded helical conformations.

NMR spectra (270MHz) are presented of strictly alternating poly(gamma-benzyl D-L-glutamate) in dioxane, chloroform, and methylene chloride solutions from which cast films have been demonstrated to have a molecular conformation in the pi pi DL 9.0 and pi pi DL 7.2 double-helical forms. Unusually low chemical shift values are observed for the alpha-CH (approximately 5.5 ppm) and NH (approximately 9.0 ppm) resonances, well separated from those of the alpha and pi DL single helices. The aromatic protons and benzyl-CH2 resonances are found slightly upfield of those of the single helices. It is proposed that these shift values are characteristic of the double helices and that the conformation of the PBD-LG sample in these solvents is the same as in the film obtained therefrom. This conclusion is supported by NMR spectra of a solvent-induced pi pi DL 7.2 leads to pi pi DL 9.0 double helical transconformation and a similar TFA-induced double helix leads to single helix conformational transition.

Benzyl Compounds↗

PubMedDate not supplied

Helical structures of poly(D-L-peptides). A conformational energy analysis.

Conformational energy calculations are reported for a number of possible helical structures of poly(D-L-peptides): the alpha helix, two single-stranded piDL, and five double-stranded pipiDL helices. For a poly(D-alanine-L-alanine) sequence, the energies of the various helices are found to differ by less than 1 kcal/(mol residue). For some helices (especially the piDL ones) two structural variants are predicted. These variants, called "goniomers", are characterized by reversed sequences of conformational angles but have the same screw sense and similar helical parameters. A biological implication of these goniomers is suggested, and their usefulness as a critical test for energy calculations is considered.

Alanine↗

PubMedDate not supplied

13C NMR spectroscopy of alternating poly(gamma-benzyl D-L-glutamate) in alpha- and double-helical conformations.

Carbon-13 NMR spectra of alpha single-helical and pipiDL double-helical conformations of alternating poly (gamma-benzyl D-L-glutamate) are presented. In both cases, the existence of doublet for the backbone carbon atoms is consistent with D and L residues being in different conformational states when engaged in the same helix. The resonances of the Calpha atoms in both helices, which are found at identical chemical shifts, and comparison with the 1H NMR observation, favor the hypotheses that solvent molecules are located inside the helical core of the double helices.

Glutamates↗

PubMedDate not supplied

Steroid-induced spinal epidural lipomatosis: CT survey.

A new case of spinal cord compression secondary to steroid-induced epidural lipomatosis is reported in a patient treated for dermatomyositis. Computed tomography was performed before the institution of steroid therapy, during spinal cord compression, and after effective surgical treatment. The development of epidural lipomatosis does not require a preexisting lipoma and may occur in a previously normal spine.

Epidural Space↗