A cyclic peptide analogue of the loop III region of platelet-derived growth factor-BB is a synthetic antigen for the native protein.

We report the synthesis and characterization of a cyclic peptide analogue of the loop III region of platelet-derived growth factor (PDGF) B-chain sequence, cyclo(73Arg-Lys-Ile-Glu-Ile-Val-Arg-Lys-Lys81-Cys), incorporating a C-terminus cysteine residue for the conjugation to a carrier protein. The synthesis involved solid-phase chemistry, utilizing Fmoc-tBu chemistry and acid labile side-chain protecting groups, followed by 'head-to-tail' cyclization using the allyl-protected glutamic acid anchored on its side chain to the solid support with HATU/HOAt as the coupling agent. Conformational differences between the cyclic and its linear counterpart PDGF peptides were determined by circular dichroism measurements in aqueous media. High titre antisera were raised to both cyclic and linear peptide immunogens. Antisera raised to the cyclic peptide cross-reacted with PDGF-BB in both Western blot and ELISA, whereas antisera raised to the linear peptide had no reactivity with PDGF-BB. The cyclic peptide (conformational design analogue) produces an immunogen which is able to antigenically mimic the secondary structure of loop III of PDGF-BB and forms a basis from which further small molecular mimetics of PDGF may be designed for use as both immunogens and also potential agonists/antagonists of PDGF. Similarly constructed immunogens may also be useful in the design of vaccines which direct responses to loop regions in other target proteins.

3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method.

The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-l-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), alpha1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0. 12+/-0.01 microM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085+/-0.04 and 0. 03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.

Raised levels of maternal serum secretory acetylcholinesterase may be indicative of fetal neural tube defects in early pregnancy.

BACKGROUND: To investigate the levels of maternal serum secretory acetylcholinesterase from a sample of pregnancies involving fetal neural tube defects and compare those results with alphafetoprotein levels. METHODS: Secretory acetylcholinesterase levels were measured using a new Enzyme Capture Immunoassay, in a small blind prospective study. The study group comprised pregnancies covering a gestational age range of 13-24 weeks where 98 had normal fetuses, 21 suffered from neural tube defects, and 15 had other complications. RESULTS: Maternal serum secretory acetylcholinesterase levels were found to be low and independent of gestational age between 14-20 weeks in a sample of normal pregnancies with normal alphafetoprotein levels. Raised levels of maternal serum secretory acetylcholinesterase were found in 100% of pregnancies involving spina bifida (17/17) and three of four anencephalics compared with raised alphafetoprotein levels found in 88% (15/17), and 100% (4/4) of the same samples. Only seven of 13 maternal serum samples from pregnancies with a normal outcome and none of the four twin pregnancies, all with raised alphafetoprotein levels, had raised secretory acetylcholinesterase levels. CONCLUSIONS: Raised levels of maternal serum secretory acetylcholinesterase may provide a useful indicator of neural tube defects in early pregnancy.

Identification of a cyclic peptide inhibitor of platelet-derived growth factor-BB receptor-binding and mitogen-induced DNA synthesis in human fibroblasts.

Peptides corresponding to residues from Loops I and III of platelet-derived growth factor-BB (PDGF-BB) were examined for their potential to act as PDGF antagonists. We have identified two peptides which directly stimulated DNA synthesis in human dermal fibroblasts and a cyclic peptide which inhibited PDGF-induced DNA synthesis. The inhibitory action of cyclic PDGF-BB(73-81), on DNA synthesis was shown to be restricted to cells which express PDGF receptors. Also cyclic PDGF-BB(73-81) specifically competed for 125I-labelled PDGF-BB but not for 125I-labelled EGF binding to their respective cellular receptors. The cyclic peptide therefore provides a minimum structure to investigate PDGF/receptor interactions and our findings confirm the importance of the loop configuration of PDGF-BB(73-81) in the native molecule. The cyclic peptide may constitute a basis for developing more potent inhibitors of PDGF action.

A cyclic peptide analogue of loop III of PDGF-BB causes apoptosis in human fibroblasts.

A cyclic peptide analogue of platelet-derived growth factor-BB (PDGF-BB), P1 [77IVRKK81-C-73RKIE76], has recently been shown to inhibit specifically [125I]PDGF-BB/receptor binding, and PDGF-BB-induced DNA synthesis in cells expressing PDGF receptors. Here we demonstrate that P1 induces apoptosis in exponentially growing human fibroblasts as confirmed by characteristic changes in cell and nuclear morphology, by TUNEL staining and by flow cytometry. Following incubation with P1 (100 microM), the percentage of cells exhibiting DNA fragmentation increased from 24% after 8 h to 76% after 28 h as exponentially growing cells progressed through the cell cycle. We conclude from these findings taken together that apoptosis accounts for the major proportion of P1-induced cell death. Omission of the Cys residue from P1 or replacement by Ser did not alter the potency of the peptide confirming that peptide dimerisation is not important for its activity. PDGF-BB, EGF, FGF, thrombin and foetal bovine serum were not able to rescue cells from the effects of P1. P1 is a useful tool for investigation of the balance of cellular proliferation/apoptosis in wound healing, atherosclerosis and restenosis, and constitutes a basis from which to design compounds with greater potency.

Synthesis, conformational properties, and antibody recognition of peptides containing beta-turn mimetics based on alpha-alkylproline derivatives.

Peptide recognition by monoclonal antibodies may provide a useful model for drug development, in particular to test the effects of conformational restriction on ligand binding. We have tested the influence of novel peptide mimetics upon conformation and binding affinity for the case of monoclonal antibodies raised to a peptide antigen which displays a preference for a beta-turn conformation in aqueous solution. Two monoclonals were isolated that recognized the peptide Ac-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala specifically at the beta-turn formed by Tyr-Pro-Tyr-Asp. Peptide analogues were then synthesized containing mimetics designed to stabilize this conformation. One, analogue (3), contained a spirocyclic gamma-lactam bridge between the alpha-position of proline-2 and the N atom of tyrosine-3, while another (2) contained (S)-alpha-methylproline at position 2. NMR spectroscopy and molecular modeling suggest that both analogues adopt reverse-turn conformations stabilized relative to that in the native sequence. For the (S)-alpha-methylproline analogue binding to both monoclonal antibodies was substantially improved, compared with the native antigen, whereas the gamma-lactam analogue (3) was not recognized by either antibody. Quantitative equilibrium ultrafiltration binding assays showed that the affinities of the (S)-alpha-methylproline analogue (2) for the two antibodies were improved over those measured with the native antigen by -2.3 and -0.65 kcal/mol. The origins of these free energy differences cannot be explained wholly on the basis of presumed extra hydrophobic contacts between the new methyl substituent and the antigen binding sites. We propose that the increased conformational stability of the analogue plays a decisive role, implying that the reverse turn detected in the native antigen, possibly a type-I turn, is important for recognition by the two antibodies.

Bispecific F(ab' gamma)2 antibody for the delivery of saporin in the treatment of lymphoma.

This work describes the successful use of bispecific F(ab' gamma)2 antibody (Ab) in combination with a ribosome-inactivating protein (RIP), saporin, for the treatment of neoplastic disease in vivo. A total of three thioether-linked F(ab' gamma)2 heterodimers were prepared, each having dual specificities for saporin and the guinea pig lymphoblastic leukemia, L2C. In all three cases specificity for the L2C cells was provided by a high affinity mouse anti-idiotype (anti-Id) mAb, whereas the antisaporin activity came from either one of two mouse mAb or an affinity-purified rabbit polyclonal Ab. In vitro studies, measuring protein synthesis, showed that all three derivatives were extremely efficient at delivering saporin to L2C cells, to the extent that addition of the rabbit Fab' gamma-containing bispecific Ab to cell culture at 1 microgram/ml increased the toxicity of saporin (50% inhibiting concentration) by close to 90,000-fold. Similarly, in leukemic guinea pigs, a small dose of saporin (10 micrograms) which by itself showed no therapeutic effect, was able to completely eradicate Id-positive tumor when given in combination with an excess of bispecific Ab. Although tumors did eventually emerge in most of these animals, immunofluorescence analysis showed that in almost all instances the escaping cells were Id- variants of the L2C. Experiments to define the optimal treatment regimen in this model showed that, although the administration of saporin and bispecific Ab at separate sites could be therapeutically effective, mixing the Ab and saporin to form immune complexes before injection did generally enhance their performance. A molar surplus of bispecific Ab in these mixtures both extended the metabolic survival of the saporin and enhanced the therapeutic performance, with molar ratios above 3:1 generally being required for optimum treatment when using saporin at 10 micrograms. Derivatives containing polyclonal antisaporin were more efficient than those containing mAb, yielding optimum therapeutic results with a molar ratio of 1.5:1 when combined with 10 micrograms saporin. These findings have shown that bispecific F(ab' gamma)2 Ab, as well as being straightforward to prepare, can also function as an extremely efficient vector for delivering cytotoxic agents such as ribosome-inactivating protein to unwanted cells in vivo.

A comparison of ELISA screening methods for the production of monoclonal antibodies against soluble protein antigens.

A library of monoclonal antibodies against pig heart citrate synthase has been raised. Twelve solid-phase immunoassay systems, employing different methods of antigen immobilization, have been compared for their ability to detect the various members of this library. It was found that a sandwich immunoassay, in which the citrate synthase antigen was immobilized on the solid support via a polyclonal antibody preparation, was the only system capable of detecting all the monoclonal antibodies tested. It is suggested that such a sandwich assay should be used in preference to direct assays for the initial screening of monoclonal antibodies.

Antibodies to nRNP, Sm, Ro(SSA) and La(SSB) detected by ELISA: their specificity and inter-relations in connective tissue disease sera.

The profile of autoantibodies to four soluble cellular ribonucleoproteins nRNP, Sm Ro(SSA) and La(SSB) was determined using ELISA with immuno-affinity-purified antigens in a connective tissue disease population. Compared with immunodiffusion, results using ELISA showed greater sensitivity but lower specificity and low titres of antibodies were frequently found in the sera of patients with connective tissue diseases other than systemic lupus erythematosus. This was true even for antibodies to Sm which have been considered highly specific for SLE. Antibodies to these antigens were predominantly of the IgG class and were capable of fixing complement irrespective of the clinical context. As previously demonstrated by immunodiffusion strong associations between anti-nRNP and anti-Sm and between anti-Ro(SSA) and anti-La(SSB) were detected by ELISA, while antibodies to nRNP and to Ro(SSA) identify distinctive serological groups. The observation that certain antibodies are closely linked suggests a relationship between the immune responses to particular antigens, and this might be explained by biological links between the antigens.