Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R.

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two non-native IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Inhibition of Siah ubiquitin ligase function.

Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1alpha (Hif-1alpha), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1alpha by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1alpha protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.

Pathotyping isolates of Newcastle disease virus using antipeptide antibodies to pathotype-specific regions of their fusion and hemagglutinin-neuraminidase proteins.

Antipeptide antibodies have been evaluated for their abilities to predict the characteristics of the cleavage motifs of the fusion protein precursors (F0) of 25 isolates of Newcastle disease virus (NDV) with a range of virulences, grouped into 12 sets according to their monoclonal antibody reactivities. A Western blot format was used to show that antisera to synthetic peptides representing sequences at the C-termini of the F2-polypeptides of defined pathotypes of NDV usually distinguish between pathotypes on the basis of their Fo cleavage sequences. However, exceptions were found with three groups of virulent isolates. Protein sequencing and mass spectral analysis of the F2-polypeptide of isolate Texas GB from one of these groups, identified an anomalous cleavage/activation process which removed the amino acids required for recognition by the antisera. This probably also explained the lack of reactivity of the Roakin isolate and low reactivity of the Komarov isolate from this group. The other exceptions involved isolates in groups with cleavage region variations from the usual motif of virulent isolates or isolates with undefined cleavage motifs. Antipeptide antisera were also raised to sections of the 45 residue C-terminal extension the hemagglutinin-neuraminidase precursor (HN0) encoded by the genes of some avirulent isolates. Western blot analysis showed that positive reactions with antibodies to peptides based on sequences between residues 577 and 613 of the HN0 was evidence for the presence of an avirulent isolate but did not exclude the presence of other pathotypes. Antisera designed to target residues 569-577 detected HN0 extensions of 6 residues on isolates known to encode such extensions. These antisera also enabled differentiation of isolates with HN0 extensions of 6 residues from those with no extension, however, it was not possible to determine the virulence of isolates based on reaction with these antisera.

Fine mapping of a surface-accessible, immunodominant site on the bluetongue virus major core protein VP7.

The 349-amino-acid major core protein VP7 of bluetongue virus (BTV) is both the most abundant viral structural protein and the major immunogenic serogroup-reactive viral antigen. Previous studies indicated that a conformation-dependent antigenic site, defined by the VP7-specific monoclonal antibody 20E9/B7/G2(20E9), was accessible from the virus surface and that the binding of the monoclonal antibody to this epitope could be blocked specifically by antisera raised against different serotypes of bluetongue virus, suggesting it is a serogroup-specific immunodominant epitope. Using a combination of three different mapping strategies, we have located the 20E9 binding site at the N-terminus of the molecule, between amino acids 30 and 48. The fine mapping of the 20E9 immunodominant epitope will facilitate structure-function analyses of the major core protein and provide new opportunities to improve existing BTV serodiagnosis methods based on this immunogenic site.

Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells.

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg-Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.

Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro.

Small cell lung cancer (SCLC) produces several neuroendocrine peptides, including gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin. There is some evidence to support the suggestion that GRP is an autocrine regulator of SCLC growth. Therefore, we have tested the effect of bombesin and two antagonists of bombesin on SCLC cell growth in a serum-free liquid tissue culture system. The antagonists used were analogues of substance P: spantide and (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. The cell lines used in this study all produced GRP-related peptides and one line had demonstrable GRP receptors. Exogenous bombesin did not cause any stimulation of growth in the liquid culture assay. The bombesin antagonists inhibited SCLC cell growth, but apparently not via the bombesin receptor. The bombesin used was biologically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bombesin-induced proliferation, which was reversed by addition of excess bombesin. In addition, the antagonists and substance P alone stimulated proliferation of 3T3 cells, indicating that they may interact with another growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because bombesin failed to stimulate growth in liquid cultures and the growth inhibition caused by bombesin antagonists was probably not mediated by the bombesin receptor.

Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin.

A gastrin binding protein (GBP) has been identified in detergent extracts of porcine gastric mucosal membranes by covalent cross-linking to 125I-[Nle15]gastrin with disuccinimidyl suberate. The apparent molecular weight of the cross-linked complex (80,000) is uneffected by reduction suggesting that the GBP is not composed of disulfide-bonded subunits. Subtraction of the molecular weight of 125I-gastrin indicates that the molecular weight of the GBP is 78,000. A similar molecular weight has been observed previously for the gastrin receptor (74,000) on intact canine parietal cells and plasma membranes therefrom, and for the receptor for the related hormone cholecystokinin (76,000-85,000) on pancreatic acinar membranes under reducing conditions. The similarity in molecular weight between the gastrin receptor and the solubilized GBP suggests that the latter protein is probably the gastrin receptor. However, the concentration (2 microM) of [Nle15]gastrin required for 50% inhibition of cross-linking of gastrin to the GBP solubilized in 0.1% Triton X-100 is 200-fold greater than the value (10 nM) observed for the gastrin receptor on isolated canine gastric parietal cells. A lower concentration (0.3 microM) of [Nle15]gastrin was required to inhibit cross-linking in a milder detergent (0.4% digitonin, 0.08% cholate). Thus, the reduced affinity for gastrin of the putative solubilized form of the gastrin receptor appears to be a result of detergent extraction.

Antigenic repeat structures in proteins of Plasmodium falciparum.

The majority of malaria antigens that have been cloned contain short sequence repeats which encode antigenic epitopes that are naturally immunogenic. Synthetic peptides have been used to show that natural antibody responses to a strain-specific Plasmodium falciparum S antigen are largely directed against epitopes encoded in an 11-amino acid sequence that is repeated approximately 100 times in the molecule. A 16-amino acid peptide conjugated to bovine serum albumin induced antibodies specific for the S antigen of the homologous isolate. Synthetic peptides have also been used to confirm the natural immunogenicity of epitopes encoded within two blocks of related repeats in the Ring-infected Erythrocyte Surface Antigen (RESA). A 16-amino acid peptide, comprising four repeats of the tetrameric sequence EENV, induced antibodies reactive with the native molecule. Detailed analyses of these anti-peptide antisera indicate that short sequence repeats express more than one epitope, some of which may cross-react with other repeat structures.

Sequence of a cDNA encoding a small polymorphic histidine- and alanine-rich protein from Plasmodium falciparum.

We describe the expression in Escherichia coli, isolation by immunological screening and complete nucleotide sequence of a cDNA clone from the malaria parasite Plasmodium falciparum. The deduced amino acid sequence contains separate blocks of repetitive hexapeptide and pentapeptide sequences and we have confirmed that these represent epitopes by reaction of the corresponding synthetic peptides with human antibodies. As the predicted size is Mr 21,000 and the overall composition is 30% His and 29% Ala, the polypeptide has been termed the small histidine-alanine rich protein (SHARP). This polypeptide is highly polymorphic in different P. falciparum isolates and cross reacts immunologically with a distinct gene product of P. falciparum. Although it is related to the Histidine Rich Protein (HRP) of P. lophurae by virtue of its high His content, it shows no obvious sequence relationship to the HRP outside the repeats.

Total synthesis of a human leukocyte interferon gene.

A 514-base pair fragment of double-stranded DNA coding for human interferon-alpha 1 (166 amino acid residues), and containing initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion, has been totally synthesized. The synthesis involved preparation of 66 oligodeoxyribonucleotides, ranging in size from 14 to 21 residues, plus 1 deoxydecanucleotide, by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. After ligation of the synthetic gene to a plasmid vector and transformation of Escherichia coli, clones containing the anticipated gene sequence were obtained.