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The incidence of papillary thyroid cancer has increased these last decades due to a better detection. High prevalence of nodules combined with the low incidence of thyroid cancers constitutes an important diagnostic challenge. We propose to develop an alternative diagnostic method to reduce the number of useless and painful thyroidectomies using a vectorized contrast agent for magnetic resonance imaging. Galectin-1 (gal-1), a protein overexpressed in well-differentiated thyroid cancer, has been targeted with a randomized linear 12-mer peptide library using the phage display technique. Selected peptides have been conjugated to ultrasmall superparamagnetic particles of iron oxide (USPIO). Peptides and their corresponding contrast agents have been tested in vitro for their specific binding and toxicity. Two peptides (P1 and P7) were selected according to their affinity toward gal-1. Their binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and they were co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Both peptides induce a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptides preserved their affinity toward gal-1. Their specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by their ability to compete with anti-gal-1 antibody. The peptides and their USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agents are potential imaging probes for thyroid cancer diagnosis. Moreover, the two gal-1-targeted peptides prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1.

The interaction between two peptides previously selected by phage display to target apoptotic cells and phospholipidic models of these cells (liposomes or micelles made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS, phosphatidylserine analog) was studied by the simple analysis of the changes induced on the proton NMR chemical shifts of the peptides. Our approach which does not need healthy and/or apoptotic cells for assessing the affinity of different peptides is fast and efficient and requires small amounts of peptide to determine the association constant, the interacting protons, and the number of interaction sites. The micellar model gave more reliable results than the liposomal one. The preferential interaction of the peptide with DPPS was evidenced by the change of the chemical shifts of specific amino acids of the peptides. Our micellar model is thus well suited to mimic apoptotic cells.

The NMR diffusometry technique, based on the measurement of the diffusion coefficient of a ligand in the absence and in the presence of its macromolecular partner, was used to study the affinity for human serum albumin (HSA) of four gadolinium complexes, potential or already used magnetic resonance imaging contrast agents. Diamagnetic lanthanum(III) ion or europium(III) ion, which has the advantage of shifting the NMR signals far away from those of the macromolecule, was used to avoid the excessive broadening of the NMR signals induced by the gadolinium(III) ion. Titration experiments, in which the HSA concentration was kept constant and the concentration of the europium or lanthanum chelate was varied, were performed to evaluate the association constant and the number of binding sites. Some additional information about the kinetics of the exchange between the free and the bound chelate was also obtained. Competition experiments with ibuprofen and salicylate, which are ligands with a known affinity for the macromolecule and for which the binding site is known, were also performed to get information about the binding site of the contrast agents.

Three techniques, electrospray mass spectrometry, ultrafiltration, and proton relaxometry, are compared in the context of the quantitative analysis of non-covalent binding between human serum albumin (HSA) and MRI contrast agents. The study of the affinity by proton relaxometry reveals the association constant and the number of interaction sites assuming that all sites are identical and independent. Ultrafiltration was adapted for the study of paramagnetic complexes. This technique confirmed the results obtained by relaxometry. Electrospray mass spectrometry, an original method able to study non-covalent binding because of its soft ionization process that allows for the survival of weak binding, provides qualitative and quantitative results. Electrospray mass spectrometry confirmed the affinity measured by proton relaxometry and ultrafiltration. This technique requires very small amounts of products and directly gives the stoichiometry of the association, information not easily obtained by classic techniques. Nevertheless, proton relaxometry remains a useful and mandatory technique for determining the enhancement of the relaxation subsequent to the binding although it demands large amounts of compounds. It is to be pointed out that even if the three techniques lead to a similar ranking of the affinity of the contrast agents for HSA, the absolute values of the association constants disagree as a result of the difference in the experimental conditions (presence of salt, native protein or desalted one, approximations in the fitting of the data, liquid or gas phases).

Among other factors influencing the residence time of the coordinated water (tauM) of paramagnetic contrast agents, the steric hindrance around the gadolinium ion seems to play a beneficial role. Such a crowding can be achieved by substituting the Gd-DTPA backbone on the C4 position. Several Gd-DTPA complexes carrying diverse groups at this position have thus been synthesised and characterised: GdS-C4-Me-DTPA, GdS-C4-n-Bu-DTPA, GdS-C4-iBu-DTPA, GdS-C4-iPr-DTPA, and Gd-C4-diMe-DTPA. TauM has been measured through the evolution of the water oxygen-17 transverse relaxation rate as a function of the temperature. The data show a reduction of tauM of GdS-C4-Me-DTPA, GdS-C4-n-Bu-DTPA, GdS-C4-iBu-DTPA, GdS-C4-iPr-DTPA, and Gd-C4-diMe-DTPA (tauM310 = 91,82, 108,98, and 57 ns respectively, as compared to Gd-DTPA (tauM310 = 143 ns)). At 310 K, the nuclear magnetic dispersion relaxation profiles of water protons are very similar for the five complexes which present longitudinal relaxivities slightly higher than those of Gd-DTPA. Regarding zinc transmetallation, C4-monosubstituted derivatives are more stable than Gd-DTPA. These results confirm that a judicious substitution of the DTPA skeleton allows for an acceleration of the coordinated water exchange rate. This observation can be useful for the design of vectorised contrast agents for molecular imaging., (Copyright 2004 ESMRMB)

Human serum albumin (HSA) binds numerous molecules, among which are suitably designed MRI contrast agents. The rotational tumbling of the protein is thus one of the parameters likely to affect the in vivo relaxivity of these agents. Literature unveils discrepancies about the value of the rotational correlation time (tau(R)) of HSA. In the present work, the tau(R) of this protein has been determined by studying the deuterium relaxation rate of small molecules known for their strong binding to HSA (warfarin and 4-hydroxycoumarin). Values of approx. 20-22 ns are obtained at 310 K in a 4% HSA solution and are in good agreement with the theoretical predictions.

MP-2269, the Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-L-aspartyl-lysine-derived-DTPA, is a small Gd-agent that binds non-covalently to serum albumin in vivo to assume the enhanced relaxivities associated with macromolecular agents, (due in part to increased rotational correlation time, tau(R)). To further explore the fundamental parameters that govern the dynamics of water proton relaxation enhancement by this prototypical albumin-binding agent, the rotational correlation time (tau(R)) for the deuterated La(III) analog of MP-2269 has been independently measured in the presence and absence of 4% albumin using 2H-NMR approaches. The diamagnetic La(III) analog of MP-2269 was deuterated at the alpha-position of the carbonyl groups. 2H-NMR studies were conducted at 7.05T (46 MHz) and 310 degrees K on a Bruker NMR spectrometer. Spectral deconvolution permitted calculation of transverse relaxation rates, 1/T(2), from the NMR linewidths and subsequently, tau(R). The results yielded a tau(R) of the albumin bound complex of approximately 8 ns. This value is intermediate between those earlier estimated by 17O-NMR ( approximately 1 ns) and 1H-NMRD ( approximately 20-50 ns) and significantly shorter than that of albumin. The 2H-NMR study results also indicate that the exchange between free and albumin-bound forms of the La(III) analog is slow (exchange lifetimes >1 ms). This slow exchange does not affect the water residence lifetimes (tau(M) 140-280 ns).

The water proton relaxation rate enhancement of the hepatospecific Gd-(S)-EOB-DTPA (Eovist) and of its (R) isomer in aqueous solutions free of protein, in serum and in 4% human serum albumin solution, are compared. In the absence of proteins, both compounds exhibit, as expected, the same proton relaxivity, as measured by the nuclear magnetic relaxation dispersion (NMRD) profiles. In serum and albumin solution, non-covalent binding of the paramagnetic complexes to macromolecules is observed. Both isomers are likely to bind to the same site of human serum albumin, but the affinity of the (S) isomer is larger than for the (R) isomer.

Chemical quality of fine roots (<1 mm diameter) was determined over a gradient of species composition in the Mixed Mesophytic Forest Region. Ash-free nitrogen, calcium, and phosphorus concentrations of roots declined by 49, 41, and 72%, respectively, over a gradient of increasing soil acidity (pH 5.3 to 4.7). Lignin concentration was unrelated to either the vegetation gradient or any of the soil changes it encompassed; however, astringent phenolics increased by 275% over the same gradient. Trends in the chemical constituency of fine roots suggest that the production of phenolics in below-ground plant parts is increased on nutrient-poor sites. This response is best related to changes in species composition, especially increasing importancy of Quercus spp.

The chemical constituency of flowering dogwood (Cornus florida L.) and red maple (Acer rubrum L.) foliage was analyzed over a species compositional gradient to test the hypothesis that over subtle gradients of moisture and nutrient availability production of phenolic compounds will be increased on sites of greatest stress. Calcium and nitrogen concentrations declined along the gradient in both species, while phosphorus showed a significant decline only in red maple. Lignin concentrations in both species were unrelated to the vegetation gradient, but astringent phenolics increased by 156% and 159% in dogwood and red maple, respectively. The correlation between production of polyphenolds and site quality supports previous observations that under conditions of environmental stress production of many secondary compounds is increased, and suggests that this relationship is significant over subtle environmental gradients.