The angiotensin II type 1 receptor (AT1R) mediates most hypertensive actions of angiotensin II. In order to understand the molecular regulation of the AT1 receptor in normal physiology and pathophysiology, methods for sensitive and specific detection of AT1R protein are required. Here, we examined the specificity of a panel of putative anti-AT1R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunocytochemistry with kidney tissue from WT mice and genetically modified mice lacking the major murine AT1R isoform, AT1A (AT1AKO), or with combined deficiency of both the AT1A and AT1B isoforms (AT1ABKO). For the 3 antibodies tested, Western blots of protein homogenates from WT kidneys yielded distinct bands with the expected size range for AT1R. In addition, these bands appeared identical in samples from mice lacking one or both murine AT1R isoforms. Additionally, the pattern of immune histo-chemical staining in kidneys, liver and adrenal glands of WT mice was very similar to that of AT1ABKO mice completely lacking all AT1 receptors. We verified the absence of AT1R subtypes in each mouse line by: 1) quantitative PCR documenting the absence of mRNA species and, 2) functionally by assessing angiotensin II-dependent vasoconstriction, which was substantially blunted in both AT1AKOs and AT1ABKOs. Finally, these antibodies failed to detect epitope-tagged AT1AR protein over-expressed in HEK cells. We conclude that anti-AT1R antibodies available from commercial sources and commonly used in published studies exhibit non-specific binding in mouse tissue that may lead to erroneous results.

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer.

The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.