ABSTRACT
High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, beta-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.
Subject(s)
Contrast Media/pharmacology , Genes, Reporter/genetics , Magnetic Resonance Imaging/methods , Animals , Embryo, Nonmammalian/metabolism , Gadolinium/metabolism , Gene Expression , Kinetics , Lac Operon/genetics , Microscopy, Fluorescence , Models, Molecular , RNA, Messenger/metabolism , Xenopus , beta-Galactosidase/metabolismABSTRACT
Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.
Subject(s)
Cobalt/chemistry , DNA-Binding Proteins/chemistry , Protein Conformation , Zinc Fingers , Amino Acid Sequence , Binding Sites , Humans , Molecular Sequence Data , Peptides/chemistry , Protein BindingABSTRACT
BACKGROUND: Photosensitizers, such as Photofrin II or Chloroaluminum-sulfonated phthalocyanine accumulate at sites of arterial injury. We have exploited this property to develop a model of photodynamic therapy (PDT) for intimal hyperplasia. The fluorescent probe [maleylated-bovine serum albumin (mal-BSA) conjugated with Texas-red] can be selectively targeted to intimal macrophages and smooth muscle cells recruited during formation of hyperplasia via a receptor-mediated mechanism. METHODS: In this study, the photosensitizer chlorin e6 (Cle6) was conjugated to mal-BSA in a rat model of intimal hyperplasia, then tested the efficacy of the ligand conjugation to photosensitizer (mal-BSA/Cle6) in PDT of intimal hyperplasia. Arterial wall injury was produced by a balloon catheter pulled through the abdominal aorta of the rat to create a model of intimal hyperplasia. Fluorescent compounds were injected two weeks after injury. RESULTS: Four hours after injection, the intensity of fluorescence achieved with injection of mal-BSA/Cle6 was higher for intimal hyperplastic lesions as compared to control areas. BSA-Cle6 unconjugated did not demonstrate such delivery. Two weeks after balloon injury, the injured aorta was irradiated externally with an argon pumped dye laser four hours following the photosensitizer injection. We employed two total radiant exposures: 20 J/cm2 and 40 J/cm2. Forty-eight hours after PDT, the arteries were examined histologically. Intimal hyperplastic cells were significantly reduced by PDT in the mal-BSA/Cle6 injected group (40-100%) versus the Cle6 group (0-20%). CONCLUSIONS: Mal-BSA/Cle6 is taken up efficiently by a scavenger pathway, localizes in areas of intimal hyperplasia, and functions as a photosensitizer for PDT.
