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Osteosarcoma (OSA) is an aggressive mesenchymal tumor of the bone that affects children and occurs spontaneously in dogs. Human and canine OSA share similar clinical, biological and genetic features, which make dogs an excellent comparative model to investigate the etiology and pathogenesis of OSA. Mitochondrial (mt) defects have been reported in many different cancers including OSA, although it is not known whether these defects contribute to OSA progression and metastasis. Taking a comparative approach using canine OSA cell lines and tumor tissues we investigated the effects of mtDNA content and dysfunction on OSA biology. OSA tumor tissues had low mtDNA contents compared to the matched non-tumor tissues. We observed mitochondrial heterogeneity among the OSA cell lines and the most invasive cells expressing increased levels of OSA metastasis genes contained the highest amount of mitochondrial defects (reduced mtDNA copies, mt respiration, and expression of electron transport chain proteins). While mitochondria maintain a filamentous network in healthy cells, the mitochondrial morphology in OSA cells were mostly "donut shaped", typical of "stressed" mitochondria. Moreover the expression levels of mitochondrial retrograde signaling proteins Akt1, IGF1R, hnRNPA2 and NFkB correlated with the invasiveness of the OSA cells. Furthermore, we demonstrate the causal role of mitochondrial defects in inducing the invasive phenotype by Ethidium Bromide induced-mtDNA depletion in OSA cells. Our data suggest that defects in mitochondrial genome and function are prevalent in OSA and that lower mtDNA content is associated with higher tumor cell invasiveness. We propose that mt defects in OSA might serve as a prognostic biomarker and a target for therapeutic intervention in OSA patients.

Three-phase partitioning (TPP) is a novel separation process used for the extraction and purification of biomolecules. The biomolecules are recovered in a purified form at the interface (precipitate), while the contaminants partition in t-butanol and aqueous phases. Peroxidase from the leaves of Ipomoea palmata was purified by using TPP. The ratio of the crude extract to t-butanol of 1:1 and 30% ammonium sulfate at 37 degrees C resulted in about 160% activity recovery and twofold purification in the aqueous phase of the first cycle of TPP. On subjecting the aqueous phase to the second cycle of TPP, a purification of 18-fold was achieved with about 81% activity recovery. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed substantial purification, and the molecular weight of peroxidase was found to be 20.1 KDa. The present study shows a higher degree of purification and activity yield as a primary purification process in comparison with existing literature values, thus demonstrating TPP as an attractive downstream process for the purification of peroxidase.

Adapt 78 (DSCR 1/calcipressin/MCIP 1) is a potent natural inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium. We previously reported two major cytosolic isoforms (1 and 4) of Adapt 78, and that isoform 4 is an oxidative and calcium stress-response protein. Using a higher cell culture density and new antibody, we again observed that both major isoforms localized to the cytosol, but a significant level of isoform 4 (but not isoform 1) was also detected in the nucleus where it was present in the non-soluble region and not associated with RNA. Exposure of cells to hydrogen peroxide led to the significant loss of isoform 4 from the nucleus with a moderate increase in cytosolic localization. The change in isoform 4 phosphorylation state in response to oxidative stress, characterized by a loss of the lesser (hypo) phosphorylated Adapt 78, was not due to accelerated degradation, although general Adapt 78 degradation was proteosome mediated. Finally, stimulation of Jurkat and primary T-lymphocyte signaling led to isoform 4 induction. This induction was BAPTA, diphenylene iodonium, and N-acetylcysteine inhibitable, and accompanied by induction of the classic immune response mediator and calcineurin-pathway-stimulated interleukin-2. These studies reveal new redox-related activities for Adapt 78 isoform 4, which may contribute to its known calcineurin-regulating and cytoprotective activities, and further suggest that Adapt 78 plays a role in basic T-cell response.

Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies. Toward understanding natural endogenous Adapt78, we used an antibody raised against cellular Adapt78 and recently determined that endogenous Adapt78 protein, like its mRNA, is oxidative and calcium stress responsive. Here we report the identification of a second endogenous form of this protein family of 41 kDa. Subcellular fractionation of human HeLa cells revealed that in contrast to results of previous transfection studies, most endogenous Adapt78, characterized as 29 and 41 kDa electrophoretic doublets, resides in the cellular cytosol. The 41 kDa form of Adapt78 was abundant and found to exhibit many characteristics in common with the previously reported oxidative stress-responsive 29 kDa form, including hypo- and hyperphosphorylation variants, rapid loss of the hypophosphorylated form following oxidative stress, response to various kinase and phosphatase inhibitors, and localization. However, it also exhibited some unique characteristics, most notably the lack of calcium inducibility. Finally, the 29 kDa form exhibited a much shorter half-life and strong stabilization following oxidant exposure compared with the 41 kDa Adapt78 form. These data reveal the presence of a novel oxidative stress-responsive 41 kDa Adapt78 species, lend further insight into the Adapt78 family of proteins and their distribution, and challenge previous conclusions obtained using transfection protocols.

DSCR1 (adapt78) is a stress-inducible gene and cytoprotectant. Its protein product, DSCR1 (Adapt78), also referred to as MCIP1, inhibits intracellular calcineurin, a phosphatase that mediates many cellular responses to calcium. Exposure of human U251 and HeLa cells to hydrogen peroxide led to a rapid hyperphosphorylation of DSCR1 (Adapt78). Inhibitor and agonist studies revealed that a broad range of kinases were not responsible for DSCR1 (Adapt78) hyperphosphorylation, including ERK1/2, although parallel activation of the latter was observed. Phosphorylation of both DSCR1 (Adapt78) and ERK1/2 was attenuated by inhibitors of tyrosine phosphatase, suggesting the common upstream involvement of tyrosine dephosphorylation. The hyperphosphorylation electrophoretic shift in DSCR1 (Adapt78) mobility was also observed with other oxidizing agents (peroxynitrite and menadione) but not nonoxidants. Calcium ionophores strongly induced the levels of both hypo- and hyper-phosphorylated DSCR1 (Adapt78) but did not alter phosphorylation status. Calcium-dependent growth factor- and angiotensin II-stimulation also induced both DSCR1 (Adapt78) species. Phosphorylation of either or both serines in a 13-amino acid peptide made to a calcineurin-interacting conserved region of DSCR1 (Adapt78) attenuated inhibition of calcineurin. These data indicate that DSCR1 (Adapt78) protein is a novel, early stage oxidative stress-activated phosphorylation target and newly identified calcium-inducible protein, and suggest that these response mechanisms may contribute to the known cytoprotective and calcineurin-inhibitory activities of DSCR1 (Adapt78).

The slow rate of demixing is a major limitation in wide commercial exploitation of aqueous two-phase systems. In the present work, use of a microwave field has been explored for the first time to enhance phase demixing rates (decrease demixing times) of these systems. The microwave-field-assisted demixing process decreased the demixing time by about 2- to 4-fold in a polyethylene glycol/potassium phosphate system and by about 1.5- to 6.5-fold in a polyethylene glycol/maltodextrin system. The enhanced demixing rate can be explained by the dipole rotation, electrophoretic migration of free salts, multiple reflections at the interfaces, droplet-droplet collision, and reduced viscosity of the continuous phase that occur during the application of a microwave field.