Differing biophysical properties underpin the unique signaling potentials within the plant phytochrome photoreceptor families.

Many aspects of photoperception by plants and microorganisms are initiated by the phytochrome (Phy) family of photoreceptors that detect light through interconversion between red light- (Pr) and far-red light-absorbing (Pfr) states. Plants synthesize a small family of Phy isoforms (PhyA to PhyE) that collectively regulate photomorphogenesis and temperature perception through redundant and unique actions. While the selective roles of these isoforms have been partially attributed to their differing abundances, expression patterns, affinities for downstream partners, and turnover rates, we show here from analysis of recombinant Arabidopsis chromoproteins that the Phy isoforms also display distinct biophysical properties. Included are a hypsochromic shift in the Pr absorption for PhyC and varying rates of Pfr to Pr thermal reversion, part of which can be attributed to the core photosensory module in each. Most strikingly, PhyB combines strong temperature dependence of thermal reversion with an order-of-magnitude faster rate to likely serve as the main physiological thermosensor, whereby thermal reversion competes with photoconversion. In addition, comparisons of Pfr occupancies for PhyA and PhyB under a range of red- and white-light fluence rates imply that low-light environments are effectively sensed by PhyA, while high-light environments, such as full sun, are effectively sensed by PhyB. Parallel analyses of the Phy isoforms from potato and maize showed that the unique features within the Arabidopsis family are conserved, thus indicating that the distinct biophysical properties among plant Phy isoforms emerged early in Phy evolution, likely to enable full interrogation of their light and temperature environments.

The biodistribution of [153Gd]Gd-labeled magnetic resonance contrast agents in a transgenic mouse model of renal failure differs greatly from control mice.

Nephrogenic systemic fibrosis occurs in renally impaired patients who have undergone contrast enhanced MR examination using intravenous gadolinium-based contrast agents. The effect of impaired kidney function on the biodistribution of gadolinium-based contrast agents was investigated using radiolabeled (153/Nat) gadolinium-DOTA, (153/Nat) gadolinium-DTPA, and (153/Nat) gadolinium-DTPA-BMA in a transgenic mouse model of renal impairment. Renally impaired animals had more activity associated with their tissues than did control mice, and this increase varied according to the radiotracer injected. For example, after 7 days, renally impaired animals that received (153/Nat) Gd-DOTA had 3-fold (P < 0.037) more activity in their bone tissue, whereas renally impaired animals receiving (153/Nat) Gd-DTPA and (153/Nat) Gd-DTPA-BMA had 8-fold (P < 0.0001) and 24-fold (P < 0.0001) more activity in their bone tissue, respectively. These findings demonstrate that renal impairment dramatically alters the tissue distribution of Gd(3+) ions in vivo, which are likely a critical factor in the development of nephrogenic systemic fibrosis.

Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

UNLABELLED: Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. METHODS: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed. RESULTS: The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. CONCLUSION: The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor-positive tumors.

Integrin α(v)ß3 as a PET imaging biomarker for osteoclast number in mouse models of negative and positive osteoclast regulation.

PURPOSE: The goal of this study was to determine the specificity of 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis. PROCEDURES: C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15 days or RANKL for 11 days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using 64Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify 64Cu-RGD in bones and other tissues. RESULTS: Mice treated with OPG showed decreased bone uptake of 64Cu-RGD at 1, 2, and 24 h post-injection of the tracer (p < 0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2 h post-injection of (64Cu-RGD (p < 0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology. CONCLUSIONS: These data demonstrate that 64Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of 64Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.

In vitro and in vivo evaluation of 64Cu-labeled SarAr-bombesin analogs in gastrin-releasing peptide receptor-expressing prostate cancer.

UNLABELLED: Bombesin is a 14-amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with (64)Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin(7-14), radiolabel the conjugate with (64)Cu, and evaluate in vitro and in vivo. METHODS: SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and (125)I-Tyr(4)-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with (64)Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. RESULTS: The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with (64)Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported (64)Cu-labeled bombesin analogs. The (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than (64)Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h). CONCLUSION: These studies demonstrate that (64)Cu-SarAr-SA-Aoc-bombesin(7-14) and (64)Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

Receptor-binding, biodistribution, and metabolism studies of 64Cu-DOTA-cetuximab, a PET-imaging agent for epidermal growth-factor receptor-positive tumors.

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.