Rhodium-Catalyzed Intermolecular Carbonylative [2 + 2 + 1] Cycloaddition of Alkynes Using Alcohol as the Carbon Monoxide Source for the Formation of Cyclopentenones

A highly regioselective rhodium-catalyzed intermolecular carbonylative [2 + 2 + 1] cycloaddition of alkynes using alcohol as a CO surrogate to access 4-methylene-2-cyclopenten-1-ones has been developed. In this transformation, the alcohol performs multiple roles, including generating the Rh–H intermediate, functioning as the CO source, and assisting in the isomerization of the alkyne. Alkynes can act as both the olefin and the alkyne partner in the cyclopentenone core

Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

<div><p>Purpose</p><p>Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.</p><p>Methods</p><p>HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.</p><p>Results</p><p>cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.</p><p>Conclusions</p><p>These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.</p></div

Enhancement of red fluorescence from cAMP treated HeLa-hNIS/tdTomato cells in mouse.

<p>(A) HeLa-hNIS/tdTomato cells (1x10⁵) were treated with cAMP (0–100 μM) at the indicated concentrations. Red fluorescent signals of cell pellets were imaged and measured using Maestro fluorescence imaging system. (B) The red fluorescent intensity that represents the expression of hNIS/tdTomato protein increased in proportion to the concentration of cAMP. (C) HeLa-hNIS/tdTomato cells were cultured with cAMP for 72 hr and then transplanted into the right flanks of mice. Untreated HeLa-hNIS/tdTomato cells were transplanted into the left flanks of mice. Red fluorescence of HeLa-hNIS/tdTomato was imaged by Maestro^TM. (D) Red fluorescent intensity of HeLa-hNIS/tdTomato cells was analyzed by the Maestro^TM software program. Bars represent mean ± SD (*, P<0.05; N = 3).</p

Generation of HeLa cells expressing the hNIS/tdTomato fusion gene.

<p>(A) Schematic representation of the hNIS/tdTomato fusion gene reporter construct (upper). RT-PCR (lower left) and Immunoblot analysis (lower right) showed stable expression of hNIS/tdTomato gene in HeLa cells. (B) Fluorescence microscope image shows the hNIS/tdTomato fusion protein expression in HeLa-hNIS/tdTomato cells. Cellular hNIS proteins were imaged using a time-lapse live cell imaging system (upper) and confocal microscopy (lower). (C) Red fluorescence from HeLa-hNIS/tdTomato cells increased with increasing cell number. (D) Function of hNIS/tdTomato in HeLa cells was measured by I-uptake and iodine uptake shows cell number dependency.</p

Changes of radioiodine uptake by regulation of glycosylation.

<p>(A) Glycosylation inhibitor, tunicamycin reduced radioiodine (¹²⁵I) uptake in the hNIS/tdTomato expressing cells. (B) Glycosylation activator, cAMP increased radioiodine (¹²⁵I) uptake in the hNIS/tdTomato expressing cells. Accumulation of radioiodine was measured with a gamma-counter at 24 hr and 72 hr after treatment (*, P<0.05; **, P<0.01; ***, P<0.001; N = 3).</p

Membrane translocation of hNIS protein by cAMP after inhibition of de novo protein synthesis.

<p>To inhibit cAMP-induced protein synthesis, AMD (5 ng/mL) or CHX (1 μg/ml) were pretreated 24h before treatment of 100 μM cAMP. (A) Enhanced expression of hNIS/tdTomato proteins by cAMP was visualized after blocking de novo protein synthesis. (B) Enhanced membrane localization of hNIS/tdTomato proteins by cAMP was visualized after blocking de novo protein synthesis. Red fluorescent intensity was analyzed with MetaMorph software. An arbitrary threshold that represented the cytosolic compartment was designated. Threshold intensity of fluorescence was adjusted to show membrane-localized hNIS/tdTomato protein only. Signals over or under the threshold were depicted as red or gray, respectively. (C) The upper threshold of red fluorescent intensity was measured to quantify the membrane localized hNIS/tdTomato protein. Confocal images were collected from at least three different regions of each sample. Bars represent mean ± SD (*, P<0.05; **, P<0.01; N = 3).</p

Membrane localization of glycosylated hNIS/tdTomato protein.

<p>(A) HeLa-hNIS/tdTomato cells were treated with tunicamycin (1.2 μM) or cAMP (100 μM), and the red fluorescent signals were photographed using confocal microscopy. Based on a cross-sectional analysis using fluorescence profiling of MetaMorph software, an arbitrary threshold that represented the cytosolic compartment was designated. Signals over the threshold were considered to be from the membrane compartment. (B) NIS expression was observed by immunoblot analysis with cellular protein extracts (20 μg) from tunicamycin- and cAMP-treated HeLa-hNIS/tdTomato cells. β-actin was used as an internal control. (C) NIS expression was observed by immunoblot analysis with membrane proteins isolated from cAMP-treated cells. Caveolin was used as an internal control.</p

Visualization and image-based quantification of membrane-localized hNIS/tdTomato protein.

<p>(A) HeLa-hNIS/tdTomato cells were treated with tunicamycin or cAMP at the indicated concentrations, and red fluorescent signals were photographed using confocal microscopy. Based on a cross-sectional analysis using fluorescence profiling of MetaMorph software, an arbitrary threshold that represented the cytosolic compartment was designated. Signals over or under the threshold were depicted as red or gray, respectively. At least three different regions of each sample were imaged. Confocal images were collected from at least three different regions of each sample. (B) Relative fluorescence signal intensities of hNIS/tdTomato proteins after the treatment of glycosylation inhibitor (tunicamycin). (C) Relative fluorescence signal intensities of hNIS/tdTomato proteins after the treatment of glycosylation activator (cAMP). Fluorescent signal intensities acquired from threshold images were measured for quantification of membrane-localized hNIS/tdTomato proteins after glycosylation inhibitor or activator treatment. Relative fluorescence signal intensities were calculated based on the fluorescence intensity of non-treated control. Bars represent mean ± SD (*, P<0.05; **, P<0.01; N = 3).</p

In Vivo Bioluminescence Imaging of Transplanted Mesenchymal Stem Cells as a Potential Source for Pancreatic Regeneration

Stem cell therapy has been studied intensively as a promising therapeutic strategy toward a cure for diabetes. To study the effect of mesenchymal stem cell (MSC) transplantation for pancreatic regeneration, we monitored the localization and distribution of transplanted MSCs by bioluminescence imaging in a mouse model. Bone marrow MSCs were isolated and transfected with a highly sensitive firefly luciferase reporter gene. To assess the efficiency of MSC transplantation, a partially pancreatectomized (PPx) mouse model was used. Transplanted MSCs were monitored by confocal microscopy and in vivo bioluminescence imaging. Daily blood glucose levels and glucose tolerance were measured. Insulin-secreting beta cells were immunostained, and insulin levels were measured via enzyme-linked immunosorbent assay. Bioluminescence signals were clearly detected from the transplanted MSCs in the pancreatic region regardless of injection route. However, locally injected MSCs exhibited more rapid proliferation than ductally injected MSCs. PPx mice harboring transplanted MSCs gradually recovered from impaired glucose tolerance. Although insulin secretion was not observed in MSCs, transplanted MSCs facilitate the injured pancreas to recover its function. In vivo optical imaging of transplanted MSCs using a highly sensitive luciferase reporter enables the assessment of MSC transplantation efficiency in a PPx mouse model

Internally-controlled and dynamic optical measures of functional tumor biology

Imaging defined aspects of functional tumor biology with bioluminescent reporter transgenes is a popular approach in preclinical drug development as it is sensitive, relatively high-throughput and low cost. However, the lack of internal controls subject functional bioluminescence to a number of unpredictable variables that reduce this powerful tool to semi-quantitative interpretation of large-scale effects. Here, we report the generation of sensitive and quantitative live reporters for two key measures of functional cancer biology and pharmacologic stress: the cell cycle and oxidative stress. We developed a two-colored readout, where two independent enzymes convert a common imaging substrate into spectrally distinguishable light. The signal intensity of one color is dependent upon the biological state, whereas the other color is constitutively expressed. The ratio of emitted colored light corrects the functional signal for independent procedural variables, substantially improving the robustness and interpretation of relatively low-fold changes in functional signal intensity after drug treatment. The application of these readouts in vitro is highly advantageous, as peak cell response to therapy can now be readily visualized for single or combination treatments and not simply assessed at an arbitrary and destructive timepoint. Spectral imaging in vivo can be challenging, but we also present evidence to show that the reporters can work in this context as well. Collectively, the development and validation of these internally controlled reporters allow researchers to robustly and dynamically visualize tumor cell biology in response to treatment. Given the prevalence of bioluminescence imaging, this presents significant and much needed opportunities for preclinical therapeutic development