No abstract

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are ligands for the EGF-receptor and act as mitogens for a variety of tissues. TGF-alpha, in particular, has been implicated as an autocrine growth factor for several cancer cell lines. Over the last 10 years many groups have examined the structure-function relationships in EGF/TGF-alpha in attempts to develop antagonists or agonists. In this review the results of these studies are summarised and related to the three-dimensional structure of EGF/TGF-alpha. The difficulties associated with the purification and characterisation of analogues of EGF/TGF-alpha and with the biological assays are discussed. It is clear that these difficulties have, in some cases, led to apparently contradicting results. The available binding data indicate that the receptor interaction surface for EGF/TGF-alpha might encompass one complete side of the molecule with a few strong binding determinants, in particular Arg41 and Leu47. The arginine at position 41 is the most critical residue and its full hydrogen-bonding capacity is needed for strong binding of EGF/TGF-alpha to the EGF-receptor. As this side of the molecule consists of residues from both the N- and C-terminal domain, it seems unlikely that agonists or antagonists can be developed on the basis of short peptides taken from the primary sequence. This concept is supported by the available binding and activity data.

No abstract

Dynamic 'H NMR measurements of the tetramethyl ether of p-tert-butylcalix[4]arene (2) show for the first time that all four possible conformations of one particular calix[4]arene are present, including the 1 ,balternate conformation. The thermodynamically most stable partial cone conformation readily interconverts to a cone or to a 1,3-alternate conformation; the interconversion to a 1,2-alternate conformation is much slower. The 1,2-alternate conformation of 2 is the kinetically stable conformation at the I H NMR time scale. The 1,a-alternate conformation was confirmed by comparison of its 'H NMR spectrum with that of the newly synthesized tetraethyl ether of p-tert-butylcalix[4]arene in a fixed 1,2-alternate conformation (6), of which the X-ray structure was determined. Partial rigidification of the calix[4]arene moiety in four different ways was achieved by replacing two of the methoxy groups of the tetramethyl ether 2 by ethoxy groups. The relative thermodynamic stabilities of the conformations of the calix[4]arene are influenced strongly by this relatively small change; in particular the 1,2-alternate conformation becomes much more stable. For the anti-1,3-diethyl-2,4-dimethyl ether 7b the 1,2-alternate is even the thermodynamically most stable conformation. Molecular mechanics calculations indicate that this is caused by the combined favorable effects on the electrostatic energy of the inside orientation of the methoxy groups and the relative large distance between the two ethoxy groups. The tetraethyl ether of p-tert-butylcalix[4]arene is not flexible at room temperature, but it equilibrates in solution at temperatures above 100 "C to a mixture of also all the four possible conformations.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and >95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

the neighboring phenol units. The second deprotonation is more difficult because the proton has to be abstracted from a negatively charged species and because in the resulting dianion every oxyanion can be stabilized by only one hydrogen bond. This shows in the gap between the pK, values for the first and second

No abstract