The Sodium Iodide Symporter (NIS) as an Imaging Reporter for Gene, Viral, and Cell-based Therapies

Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter

Halothane binds to druggable sites in the [Ca2+]4-calmodulin (CaM) complex, but does not inhibit [Ca2+]4-CaM activation of kinase

The mechanism(s) of volatile anesthetics (VA) are poorly understood. We used high resolution NMR spectroscopy to determine the structure of the halothane/calmodulin([Ca2+]4-CaM) complex, and found that halothane molecules bind in the druggable sites. We then examined whether VA binding to druggable sites in calmodulin would effect [Ca2+]4-CaM dependent activity of myosin light chain kinase. We used fluorescence assays to determine that VA effect [Ca2+]4-CaM activation of smooth-muscle-myosin-light-chain-kinase (smMLCK), but not the Kd of [Ca2+]4-CaM binding to skeletal-myosin-light-chain-kinase-peptide recognition sequence (skMLCKp). These results suggest that VA do not alter [Ca2+]4-CaM dependent MLCK activity via direct interactions with [Ca2+]4-CaM

Purification and Characterization of a Soybean Root Nodule Phosphatase Expressed in \u3ci\u3ePichia pastoris\u3c/i\u3e

Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5’-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In order to perform directed mutagenesis and more extensive biochemical characterization, a means of producing recombinant ACP was needed. Several attempts were made to express ACP in Escherichia coli, but all conditions employed resulted in protein that was found entirely in inclusion bodies, and resolubilization experiments were unsuccessful. Therefore, the methyltrophic yeast Pichia pastoris was chosen as a eukaryotic expression host. The coding sequence of ACP was cloned into the pPIC9 vector to create a fusion with the yeast α mating factor secretion signal. The ACP:pPIC9 construct was integrated into P. pastoris strain GS115. Expression of ACP was under the control of an alcohol oxidase methanol-inducible promoter. Methanol induction resulted in secretion of ACP to a level of 10 mg/L. The recombinant ACP was purified 550-fold to homogeneity by phenyl-Sepharose, hydroxyapatite, and MonoS chromatography. The purified enzyme had Km values of 0.08 and 0.12 for 5’-AMP and 5’-GMP. These values were similar to those obtained for the native ACP heterodimer purified from soybean (0.08 and 0.15 mM for 5’-AMP and 5’-GMP). The specific activity of the recombinant enzyme for all substrates tested was 1.6- to 1.8-fold higher than the values for the purified soybean heterodimer

Transcriptomic and Immunohistochemical Profiling of SLC6A14 in Pancreatic Ductal Adenocarcinoma

We used a target-centric strategy to identify transporter proteins upregulated in pancreatic ductal adenocarcinoma (PDAC) as potential targets for a functional imaging probe to complement existing anatomical imaging approaches. We performed transcriptomic profiling (microarray and RNASeq) on histologically confirmed primary PDAC tumors and normal pancreas tissue from 33 patients, including five patients whose tumors were not visible on computed tomography. Target expression was confirmed with immunohistochemistry on tissue microarrays from 94 PDAC patients. The best imaging target identified was SLC6A14 (a neutral and basic amino acid transporter). SLC6A14 was overexpressed at the transcriptional level in all patients and expressed at the protein level in 95% of PDAC tumors. Very little is known about the role of SLC6A14 in PDAC and our results demonstrate that this target merits further investigation as a candidate transporter for functional imaging of PDAC