Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis.

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.

Design and synthesis of inhibitors incorporating beta -amino acids of metalloendopeptidase EC 3.4.24.15.

Endopeptidase EC 3.4.24.15 (EP 24.15) is a thermolysin-like metalloendopeptidase which is expressed widely throughout the body, with the highest concentrations in the brain, pituitary and testis. While the precise role of EP 24.15 remains unknown, it is thought to participate in the regulated metabolism of a number of specific neuropeptides. Of the limited number of inhibitors described for EP 24.15, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but is unstable in vivo due to its cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). The cpp-Ala-Ala N-terminal product of this cleavage is a potent inhibitor of angiotensin converting enzyme, which further limits the use of CFP in vivo. To generate specific inhibitors of EP 24.15 that are resistant to in vivo proteolysis by EP 24.11, beta-amino acids have been incorporated into the structure of CFP. We have prepared racemic mixtures of beta-amino acids containing proteogenic side chains, which are 9-fluorenylmethoxycarbonyl (Fmoc)-protected, and several analogues of CFP containing beta-amino acids have been synthesized by solid phase peptide synthesis. The results of stability and inhibitory studies of these new analogues show that the incorporation of beta-amino acids adjacent to the scissile bond can indeed stabilize the peptides against cleavage by EP 24.11 and still inhibit EP 24.15. The results obtained in these studies demonstrate the potential of these amino acids in the synthesis of peptidomimetics and in the design of new stable and specific therapeutics.

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.

Tropane-based amino acids for peptide structure-function studies: inhibitors of platelet aggregation.

Novel tropane (azabicycloheptane) and azabicyclohexane containing amino acids have been prepared and incorporated into analogues of reported inhibitors of platelet aggregation. The influence of these central constraints upon biological activity suggest their utility in peptide structure function studies.