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  1. Article ; Online: MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

    Linshan Liu / Kobra Alizadeh / Sarah C Donnelly / Praveen Dassanayake / Tian Tian Hou / Rebecca McGirr / R Terry Thompson / Frank S Prato / Neil Gelman / Lisa Hoffman / Donna E Goldhawk

    PLoS ONE, Vol 14, Iss 6, p e

    2019  Volume 0217842

    Abstract: Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast ... ...

    Abstract Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2' (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.
    Keywords Medicine ; R ; Science ; Q
    Subject code 570
    Language English
    Publishing date 2019-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Characterization of 5-(2-18F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas [version 2; referees

    Ahmed Abbas / Christine Beamish / Rebecca McGirr / John Demarco / Neil Cockburn / Dawid Krokowski / Ting-Yim Lee / Michael Kovacs / Maria Hatzoglou / Savita Dhanvantari

    F1000Research, Vol

    2 approved]

    2016  Volume 5

    Abstract: Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. ...

    Abstract Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2-18F-fluoroethoxy)-L-tryptophan (18F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18F-L-FEHTP uptake with beta cell mass. Conclusions: 18F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.
    Keywords Diabetes & Obesity ; Methods for Diagnostic & Therapeutic Studies ; Pancreas ; Medicine ; R ; Science ; Q
    Language English
    Publishing date 2016-11-01T00:00:00Z
    Publisher F1000 Research Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Synthesis and Evaluation of Optical and PET GLP-1 Peptide Analogues for GLP-1R Imaging

    Babak Behnam Azad / Vanessa Rota / Lihai Yu / Rebecca Mcgirr / André H. St. Amant / Ting-Yim Lee / Savita Dhanvantari / Leonard G. Luyt PhD

    Molecular Imaging, Vol

    2015  Volume 14

    Abstract: A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out ...

    Abstract A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68 Ga-labeled GLP-1 (7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.
    Keywords Biology (General) ; QH301-705.5 ; Medical technology ; R855-855.5
    Language English
    Publishing date 2015-01-01T00:00:00Z
    Publisher Hindawi - SAGE Publishing
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes

    Douglas, Gregory A.F / Carlie L. Charlton / Dov B. Kagan / Leonard G. Luyt / Lisa M. Hoffman / Rebecca McGirr / Savita Dhanvantari

    Peptides. 2014 Apr., v. 54

    2014  

    Abstract: Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin ... ...

    Abstract Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr3(octanoyl), Lys19(Cy5)]ghrelin (1–19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys19(Cy5)]ghrelin (1–19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.
    Keywords binding capacity ; cardiomyocytes ; fluorescein ; ghrelin ; ghrelin receptors ; humans ; image analysis ; isothiocyanates ; mice
    Language English
    Dates of publication 2014-04
    Size p. 81-88.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 769028-9
    ISSN 1873-5169 ; 0196-9781
    ISSN (online) 1873-5169
    ISSN 0196-9781
    DOI 10.1016/j.peptides.2014.01.011
    Database NAL-Catalogue (AGRICOLA)

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  5. Article ; Online: Magnetic Resonance Imaging of Cells Overexpressing MagA, an Endogenous Contrast Agent for Live Cell Imaging

    Donna E. Goldhawk / Claude Lemaire / Cheryl R. McCreary / Rebecca McGirr / Savita Dhanvantari / R. Terry Thompson / Rene Figueredo / Jim Koropatnick / Paula Foster / Frank S. Prato

    Molecular Imaging, Vol

    2009  Volume 8

    Abstract: Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA , a ... ...

    Abstract Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA , a putative iron transporter. We have examined expression of MagA in mouse neuroblastoma N2A cells and characterized their response to iron loading and cellular imaging by MRI. MagA expression augmented both Prussian blue staining and the elemental iron content of N2A cells, without altering cell proliferation, in cultures grown in the presence of iron supplements. Despite evidence for iron incorporation in both MagA and a variant, MagA E137V , only MagA expression produced intracellular contrast detectable by MRI at 11 Tesla. We used this stable expression system to model a new sequence for cellular imaging with MRI, using the difference between gradient and spin echo images to distinguish cells from artifacts in the field of view. Our results show that MagA activity in mammalian cells responds to iron supplementation and functions as a contrast agent that can be deactivated by a single point mutation. We conclude that MagA is a candidate MRI reporter gene that can exploit more fully the superior resolution of MRI in noninvasive medical imaging.
    Keywords Biology (General) ; QH301-705.5 ; Medical technology ; R855-855.5
    Subject code 610
    Language English
    Publishing date 2009-05-01T00:00:00Z
    Publisher Hindawi - SAGE Publishing
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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