In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein ␣ (G␣) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G␣ i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G␣ i , thus preventing its GEF activity for G␣ i . Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G␣ i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.

Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism-jaw tumor familial cancer syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism. Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex. This study addressed the expression and functional properties of human parafibromin. A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle. Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments. Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas. Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia. These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.

Sensitive, specific, and noninvasive detection of angiogenesis would be helpful in discovering new strategies for the treatment of cardiovascular diseases. Recently, we reported the 64Cu-labeled C-type atrial natriuretic factor (CANF) fragment for detecting the upregulation of natriuretic peptide clearance receptor (NPR-C) with PET on atherosclerosis-like lesions in an animal model. However, it is unknown whether NPR-C is present and overexpressed during angiogenesis. The goal of this study was to develop a novel CANF-integrated nanoprobe to prove the presence of NPR-C and offer sensitive detection with PET during development of angiogenesis in mouse hind limb. Methods We prepared a multifunctional, core-shell nanoparticle consisting of DOTA chelators attached to a poly(methyl methacrylate) core and CANF-targeting moieties attached to poly(ethylene glycol) chain ends in the shell of the nanoparticle. Labeling of this nanoparticle with 64Cu yielded a high-specific-activity nanoprobe for PET imaging NPR-C receptor in a mouse model of hind limb ischemia–induced angiogenesis. Histology and immunohistochemistry were performed to assess angiogenesis development and NPR-C localization. Results 15O-H2O imaging showed blood flow restoration in the previously ischemic hind limb, consistent with the development of angiogenesis. The targeted DOTA-CANF-comb nanoprobe showed optimized pharmacokinetics and biodistribution. PET imaging demonstrated significantly higher tracer accumulation for the targeted DOTA-CANF-comb nanoprobe than for either the CANF peptide tracer or the nontargeted control nanoprobe (P < 0.05, both). Immunohistochemistry confirmed NPR-C upregulation in the angiogenic lesion with colocalization in both endothelial and smooth muscle cells. PET and immunohistochemistry competitive receptor blocking verified the specificity of the targeted nanoprobe to NPR-C receptor. Conclusion As evidence of its translational potential, this customized DOTA-CANF-comb nanoprobe demonstrated superiority over the CANF peptide alone for imaging NPR-C receptor in angiogenesis.

According to the International Association for the Study of Pain (IASP) pain is characterized as an “unpleasant sensory and emotional experience associated with actual or potential tissue damage”. The TRP super-family, compressing up to 28 isoforms in mammals, mediates a myriad of physiological and pathophysiological processes, pain among them. TRP channel might be constituted by similar or different TRP subunits, which will result in the formation of homomeric or heteromeric channels with distinct properties and functions. In this review we will discuss about the function of TRPs in pain, focusing on TRP channles that participate in the transduction of noxious sensation, especially TRPV1 and TRPA1, their expression in nociceptors and their sensitivity to a large number of physical and chemical stimuli.