PurposeHuman 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.MethodsHeLa 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.ResultscAMP-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.ConclusionsThese 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.
We investigated a therapeutic strategy for recurrent malignant gliomas using mesenchymal stem cells (MSC), expressing cytosine deaminase (CD), and prodrug 5-Fluorocytosine (5-FC) as a more specific and less toxic option. MSCs are emerging as a novel cell therapeutic agent with a cancer-targeting property, and CD is considered a promising enzyme in cancer gene therapy which can convert non-toxic 5-FC to toxic 5-Fluorouracil (5-FU). Therefore, use of prodrug 5-FC can minimize normal cell toxicity. Analyses of microarrays revealed that targeting DNA damage and its repair is a selectable option for gliomas after the standard chemo/radio-therapy. 5-FU is the most frequently used anti-cancer drug, which induces DNA breaks. Because dihydropyrimidine dehydrogenase (DPD) was reported to be involved in 5-FU metabolism to block DNA damage, we compared the survival rate with 5-FU treatment and the level of DPD expression in 15 different glioma cell lines. DPD-deficient cells showed higher sensitivity to 5-FU, and the regulation of DPD level by either siRNA or overexpression was directly related to the 5-FU sensitivity. For MSC/CD with 5-FC therapy, DPD-deficient cells such as U87MG, GBM28, and GBM37 showed higher sensitivity compared to DPD-high U373 cells. Effective inhibition of tumor growth was also observed in an orthotopic mouse model using DPD- deficient U87MG, indicating that DPD gene expression is indeed closely related to the efficacy of MSC/CD-mediated 5-FC therapy. Our results suggested that DPD can be used as a biomarker for selecting glioma patients who may possibly benefit from this therapy.
An interpenetrating polymer network (IPN) hydrogel based on poly(propylene glycol) and poly(acrylic acid) was prepared by UV irradiation. The swelling behavior of the IPN hydrogel was studied by the immersion of the gel in aqueous NaCl solutions of various concentrations. The swelling ratio decreased with an increase in the NaCl concentration. The electrically sensitive behavior of the IPN hydrogel in electric fields was also investigated. The IPN hydrogel also showed a stepwise bending behavior that depended on the electric stimulus. The bending angle and bending speed of the IPN hydrogel were greatest in 0.6 wt % aqueous NaCl and increased with an increase in the applied voltage.
Complexes of lanthanide with Mordant Red 19 (MR19) are shown to be adsorbed and can he electrochemically reduced at mercury electrodes at a more negative potential than where MR19 is reduced. Based on these properties, an analytical application for the determination of lanthanides is demonstrated by linear sweep voltammetry (LSV) and by highly sensitive adsorptive stripping voltammetry (AdSV). By applying AdSV, the detection limit of several lanthanides is about 5 . 0~ lo-'" M with an accumulation time of 5 min and the linear response range is from 5 . 0~ lo-' M to 1 .Ox 1 V 6 M. A typical relative standard deviation i s found to be 5.2 % at the 5 . 0~ lO-'M level. The results from LSV show that the difference in peak potentials (AQ between complex and MR19 increases when the atomic number of lanthanide ion is increased. Because the smallest AE, due to the presence ofLa3+ is still large enough to distinguish the complex peak from dye peak, it is possible to recognize all members of lanthanide ions selectively with MR19, regardless of whether they are light or heavy ones. It is also possible to perform simultaneous analysis for some mixtures. In addition, the range of lanthanide concentration, for which quantitative analysis is valid, becomes wider as a result of using LSV as well as AdSV.
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.
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