• Journal of Applied Biological Chemistry
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• v.57 no.3
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• pp.259-265
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• 2014

Mitochondrial DNA (mtDNA)-depleted (${\rho}^0$) cells are often used as mtDNA recipients to study the interaction between the nucleus and mitochondria in mammalian cells. Therefore, it is crucial to obtain mtDNA-depleted cells with many different nuclear backgrounds for the study. Here, we demonstrate a rapid and reliable method to isolate mammalian mtDNA-depleted cells involving treatment with the antimitochondrial agents ethidium bromide (EtBr) and 2',3'-dideoxycytidine (ddC). After a short exposure to EtBr or ddC, followed by rapid clonal isolation, we were able to generate viable mtDNA-depleted cells from mouse and human cells and were able to successfully repopulate them with exogenous mitochondria from platelets isolated from mouse and human blood samples. These mtDNA-depleted cells can be used to characterize the nuclear mitochondrial interactions and to study mtDNA-associated defects in mammalian cells. Our method of isolating mtDNA-depleted cells is practical and applicable to a variety of cell types.

• The Korean Journal of Pharmacology
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• v.31 no.3
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• pp.365-375
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• 1995

The anemia-inducing strain of Friend virus (FVA) is a murine retrovirus which stimulates the proliferation of erythroid progenitor cells. The progenitor cells synthesized by FVA-stimulation are unable to proceed with differentiation and accumulate in the spleen resulting in splenomegaly in infected mice. Using FVA-inoculated mice as a model, we have investigated the antiretroviral effects of 2',3'-dideoxycytidine (ddC) and recombinant $interferon-{\alpha}-A\;(rIFN-{\alpha}-A)$ on FVA infection. The extent of the infection was determined by measuring the weights of the spleens. Daily intraperitoneal injection of ddC (100 mg/kg body weight), $rIFN-{\alpha}-A$ (10 KU/mose) and the combination of both drugs to FVA inoculated mice for 18 days resulted in suppression of the growth of spleens by 15.1%, 52.7% and 61.6%, respectively. When ddC was dissolved in drinking water (0.1 mg/ml) and administered to a group of FVA inoculated mice ad libitum, and $rIFN-{\alpha}-A$ (10 KU/mouse) was intraperitoneally injected daily to another group of ddC (0.1 mg/ml) drinking mice for 18days, the growth of spleens was suppressed by 38.4% and 83.2%, respectively. These results indicate that administration of ddC via drinking water is more effective in suppressing FVA infection than the daily injection of ddC, and that the combined effects ddC and $rIFN-{\alpha}-A$ are not synergistic but additive. In order to determine whether ddC treatment alters the characteristic of the progenitor cells with respect to $Ca^{++}$ uptake, $Ca^{++}$ uptake in erythroid cells and the effect of cyclohexyladenosine (CHA) on the $Ca^{++}$ uptake were studied. $Ca^{++}$ uptake in the erythroid progenitor cells was about 20-fold greater than in mouse erythrocytes and the inhibition of $Ca^{++}$ uptake by CHA was the greatest in the progenitor cells from FVA infected mice which were treated with ddC. The inhibition was obviated by theophylline. Results of CHA binding studies showed that the erythroid progenitor cells contain both high and low affinity CHA binding sites, whereas mose erythrocytes contain only the low affinity CHA binding sites.

• Biomolecules & Therapeutics
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• v.22 no.6
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• pp.532-539
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• 2014

Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC ($50{\mu}mol/L$), gp120 (500 pmol/L) plus ddC ($50{\mu}mol/L$), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC ($50{\mu}mol/L$) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC ($50{\mu}mol/L$) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (> $25{\mu}m$), whereas ddC mainly affected small diameter DRG neurons (${\leq}25{\mu}m$). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.