[Show abstract][Hide abstract] ABSTRACT: Background
Type 2 diabetes results from failure of the β-cells to compensate for increased insulin demand due to abnormal levels of metabolic factors. The ob/ob(lep-/-) mouse has been extensively studied as an animal model of type 2 diabetes. Previous studies have shown a correlation between β-cell function and bioluminescent imaging in lean genetically engineered mice. The ability to noninvasively monitor β-cell function in ob/ob mice could provide new information on β-cell regulation in type 2 diabetes.
To create the B6 Albino ob/ob MIP-luc mice (ob/ob-luc), the ob/ob mouse was crossed with the CD1 MIP-luc mouse. All mice were backcrossed over multiple generations to ensure the genetic background of the transgenic mice was over 96% similar to the background of the original ob/ob mouse. Animal weight, blood glucose levels, insulin in plasma, and in vivo bioluminescence (BLI) were monitored weekly or biweekly for up to 70 weeks of age. BL imaging was performed using IVIS Spectrum (Perkin Elmer) and calculated by integrating the bioluminescence signal between 5 and 10 min after i.v. injection of D-luciferin. Insulin immunohistochemistry determined islet beta cell count and insulin secretion assay determined islet insulin function.
There were significant increases in BLI and insulin levels as the ob/ob-luc mice aged while glucose levels gradually decreased. Ob/ob-luc were sacrificed at different time points to determine ex vivo BLI, islet function and total β-cell numbers using a cell counting training algorithm developed for the Vectra image analysis system (Perkin Elmer). The number of β-cells increased as the mice aged and all three ex vivo measurements correlated with BLI.
The ob/ob-luc mice can serve as a model of metabolic stress, similar to human type 2 diabetes using BLI as a surrogate marker for β-cell function.
[Show abstract][Hide abstract] ABSTRACT: We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.
No preview · Article · Jun 2013 · ACS Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Purpose
Visualization of the cell cycle in living subjects has long been a big challenge. The present study aimed to noninvasively visualize mitotic arrest of the cell cycle with an optical reporter in living subjects.
An N-terminal cyclin B1–luciferase fusion construct (cyclin B-Luc) controlled by the cyclin B promoter, as a mitosis reporter, was generated. HeLa or HCT116 cells stably expressing cyclin B-Luc reporter were used to evaluate its cell cycle-dependent regulation and ubiquitination-mediated degradation. We also evaluated its feasibility to monitor the mitotic arrest caused by Taxotere both in vitro and in vivo.
We showed that the cyclin B-Luc fusion protein was regulated in a cell cycle-dependent manner and accumulated in the mitotic phase (M phase) in cellular assays. The regulation of cyclin B-Luc reporter was mediated by proteasome ubiquitination. In the present study, in vitro imaging showed that antimitotic reagents like Taxotere upregulated the reporter through cell cycle arrest in the M phase. Noninvasive longitudinal bioluminescence imaging further demonstrated an upregulation of the reporter consistent with mitotic arrest induced in tumor xenograft models. Induction of this reporter was also observed with a kinesin spindle protein inhibitor, which causes cell cycle blockage in the M phase.
Our results demonstrate that the cyclin B-Luc reporter can be used to image whether compounds are capable, in vivo, of causing an M phase arrest and/or altering cyclin B turnover. This reporter can also be potentially used in high-throughput screening efforts aimed at discovering novel molecules that will cause cell cycle arrest at the M phase in cultivated cell lines and animal models.
Full-text · Article · Feb 2013 · Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging
[Show abstract][Hide abstract] ABSTRACT: beta-Galactosidase (beta-gal) (encoded by the lacZ gene) has been widely used as a transgene reporter enzyme. The ability to image lacZ expression in living transgenic animals would further extend the use of this reporter. It has been reported that 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-beta-d-galactopyranoside (DDAOG), a conjugate of beta-galactose and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one), is not only a chromogenic lacZ substrate but that the cleavage product has far-red fluorescence properties detectable by in vivo imaging. In an attempt to noninvasively image lacZ expression in vivo, we applied fluorescence imaging to a G protein-coupled receptor (GPR56), knockout (KO) mouse model, in which the lacZ gene is introduced in the GPR56 locus. Compared to wild-type (WT) mice, GPR56KO/LacZ mice showed three- to fourfold higher fluorescence intensity in the head area 5 min after tail-vein injection of DDAOG. beta-Gal staining in sections of whole brain showed strong lacZ expression in homozygotes, but not in WT mice, consistent with lacZ activity detected by in vivo imaging. The kidneys were also visualized with fluorescence imaging both in vivo and ex vivo, consistent with beta-gal staining findings. Our results demonstrate that fluorescence imaging can be used for in vivo real-time detection of lacZ activity by fluorescence imaging in lacZ transgenic mice. Thus, this technology can potentially be used to noninvasively image changes of certain endogenous molecules and/or molecular pathways in transgenic animals.
Full-text · Article · Sep 2009 · Assay and Drug Development Technologies
[Show abstract][Hide abstract] ABSTRACT: We have applied noninvasive optical imaging technology to the in vivo hollow fiber assay, using tumor cell lines in which optical reporters are expressed in response to activation/inhibition of a specific molecular pathway. In vivo noninvasive imaging of molecular pathways in cells within hollow fibers enables a rapid and accurate evaluation of drug targets and provides useful insights to guide novel drug discovery. In this protocol we show, as an example, that a luciferase reporter, driven by the responsive element of nuclear factor NF-kappaB, was induced in cells within hollow fibers implanted in living mice, and a detailed procedure for in vivo bioluminescence imaging of hollow fibers is described. This approach can, in principle, be applied to image any molecular pathways of interest when appropriate reporter cells are generated. Hollow fiber encapsulation and implantation takes 2 d, and in vivo validation of reporter takes 1-2 weeks.
[Show abstract][Hide abstract] ABSTRACT: The in vivo hollow fiber assay, in which semipermeable hollow fibers filled with tumor cells, are implanted into animals, was originally developed to screen for anticancer compounds before assessment in more complex tumor models. To enhance screening and evaluation of anticancer drugs, we have applied optical imaging technology to this assay. To demonstrate that tumor cells inside hollow fibers can communicate with the host mice, we have used fluorescence imaging in vivo and CD31 immunostaining ex vivo to show that angiogenesis occurs around cell-filled hollow fibers by 2 weeks after subcutaneous implantation. Bioluminescence imaging has been used to follow the number of luciferase-expressing tumor cells within implanted hollow fibers; proliferation of those cells was found to be significantly inhibited by docetaxel or irinotecan. We also used bioluminescence imaging of hollow fibers to monitor the nuclear factor kappaB (NFkappaB) pathway in vivo; NFkappaB activation by lipopolysaccharide and tumor necrosis factor-alpha was evaluated in tumor cell lines genetically engineered to express luciferase controlled by an NFkappaB-responsive element. These results demonstrate that optical imaging of hollow fibers containing reporter tumor cells can be used for the rapid and accurate evaluation of antitumor activities of anticancer drugs and for measurement of molecular pathways.
Full-text · Article · Sep 2007 · Neoplasia (New York, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: We report the critical residues for the interaction of the kinins with human bradykinin receptor 1 (B1) using site-directed mutagenesis in conjunction with molecular modeling of the binding modes of the kinins in the homology model of the B1 receptor. Mutation of Lys118 in transmembrane (TM) helix 3, Ala270 in TM6, and Leu294 in TM7 causes a significant decrease in the affinity for the peptide agonists des-Arg10kallidin (KD) and des-Arg9BK but not the peptide antagonist des-Arg10Leu9KD. In contrast, mutations in TM2, TM3, TM6, and TM7 cause a significant decrease in the affinity for both the peptide agonists and the antagonist. These data indicate that the B1 bradykinin binding pocket for agonists and antagonists is similar, but the manners in which they interact with the receptor do not completely overlap. Therefore, there is a potential to influence the receptor's ligand selectivity.
[Show abstract][Hide abstract] ABSTRACT: An iterative analogue library synthesis approach rapidly delivered (S)-13 h, a potent, reversible, and selective GlyT1 inhibitor. (S)-13 h selectively increased glycine levels in the prefrontal cortex to 340% of basal levels and significantly enhanced prepulse inhibition in mice. Thereby, providing strong support for the development of novel antipsychotics based on the NMDA hypofunction hypothesis of schizophrenia.
[Show abstract][Hide abstract] ABSTRACT: We have characterized the interaction of the serotonin transporter ligand [3H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [3H]-DASB, a tritiated version of the widely used [11C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K(d)=0.20+/-0.04 nM). The serotonin transporter density (B(max)) obtained for rhesus frontal cortex was found to be 66+/-8 fmol/mg protein using [3H]-DASB, similar to the B(max) value obtained using the reference radioligand [3H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83+/-22 fmol/mg protein). Specific binding sites of both [3H]-DASB and [3H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [3H]-citalopram binding in a competition autoradiographic study, with K(i) values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [3H]-DASB and [3H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [11C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.
No preview · Article · Jun 2006 · Nuclear Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: Antagonists of the B1 bradykinin receptor (B1R), encoded by the BDKRB1 gene, offer the promise of novel therapeutic agents for inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the B1R. To circumvent these issues we generated two genetically engineered rodent models. The first is a transgenic rat over-expressing the human B1R under the control of the neuronal-specific enolase promoter; we previously reported the utility of this model in assessing human B1R receptor occupancy in the central nervous system of the rat. The second model, reported here, utilized gene-targeting by homologous recombination to replace the genomic coding sequence for the endogenous mouse B1R with that of the human B1R. The mRNA expression profile of the humanized Bdkrb1 (hBkdrb1) allele is similar to that of the mouse Bdkrb1 (mBkdrb1) in the wild-type animal. Furthermore, in vitro assays indicate that tissues isolated from the humanized mouse possess pharmacological properties characteristic of the human B1R. Therefore, we have generated a humanized B1R mouse model that is suitable for testing the efficacy of human B1R-selective compounds.
No preview · Article · Mar 2006 · Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: This study used behavioural and in vivo electrophysiological paradigms to examine the effects of systemic and spinal administration of a bradykinin B1 receptor antagonist, compound X, on acute nociceptive responses in the rat. In behavioural experiments, compound X significantly increased the latency to withdraw the hindpaw from a radiant heat source after both intravenous and intrathecal administration, without affecting motor performance on the rotarod. In electrophysiological experiments, both intravenous and direct spinal administration of compound X attenuated the responses of single dorsal horn neurones to noxious thermal stimulation of the hindpaw. These data show that the antinociceptive effects of a bradykinin B1 receptor antagonist are mediated, at least in part, at the level of the spinal cord and suggest a role for spinal bradykinin B1 receptors in acute nociception.
No preview · Article · Jan 2006 · European Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.
No preview · Article · Jun 2005 · Biochemical and Biophysical Research Communications