• 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.

• Environmental Mutagens and Carcinogens
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• v.24 no.3
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• pp.143-150
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• 2004

In mammalian, cytochrome P4501A1 (CYP1A1) is very important for metabolism of xenobiotics such as PAHs(Polycyclic aromatic hydrocarbon) and heterocyclic amine, and it is induced by environmental contaminants such as PAHs, TCDD(2,3,7,8-tetrchlorodibenzo-p-dioxin) and 3-MC (3-methylcholanthrene). In fish, like mammalian, when it is exposed to environmental contaminants, they cause specific and sensitive induction of CYP1A. Therefore, induction of CYP1A in fish and mammalian is widely used as a biomarker for exposure of environmental contaminants. In this study, to compare the function of Cyp1a1 in fish with it in mammalian, we have used rainbow trout(Oncorhynchys mykiss) hepatoma cells (RTH-149) and mouse hepatocyte (Hepa-I). in order to examine induction of Cyp1a1 by TCDD, we have used the bioassay system. We examined effects of TCDD on the Cyp1a1-luciferase reporter gene activity, 7-ethoxyresorufin O-deethylase(EROD) activity and Cypa mRNA level.

• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.183-193
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• 1999

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is synthesized as a 160 KDa precursor, gp160, that is cleaved by a cellular protease to form the gp120 and gp41 subunits. Mammalian expression vectors were designed that are capable of efficient expression of various mutant envelope glycoproteins derived from a molecular clone of HIV-1. To construct these vectors, one type of mutation was made at the gp120-gp41 cleavage site by oligonucleotide-directed mutagenesis. And another mutation was made to change amino acids in the membrane spanning region of HIV-1 gp41 important for membrane anchorage. Next, these two mutations were combined to generate a vector to have double mutations in cleavage site and membrane-spanning region. These mutants were transiently expressed in mammalian cells. The effect of these mutations on envelope glycoprotein synthesis, proteolytic processing and secretion was determined. In addition, cell surface expression and ability of the glycoprotein to induce syncytium formation were examined. This study provides a mammalian expression system that is capable of efficient expression and secretion of soluble gp160.

• Korean Journal of Biological Psychiatry
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• v.23 no.1
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• pp.18-23
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• 2016

Depression is a complicated psychiatric illness with severe consequences. Despite recent advanced achievements of molecular neurobiology, pathophysiology of depression has not been well elucidated. Among new findings of pathophysiology of depression, the possible fast antidepressant effect by N-methyl-D-asparate receptor antagonist, such as ketamine, is regarded as a promising treatment target of depression. Ketamine stimulates the mammalian target of rapamycin (mTOR) signaling pathway and activation of mTOR signaling pathway may be a key mechanism of the antidepressant effect of ketamine. Thus, this review describes the role of mTOR signaling in the pathophysiology of depression and developing a new treatment target of depression.

• Animal cells and systems
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• v.13 no.2
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• pp.119-125
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• 2009

Mammalian hibernation is a type of natural adaptation that allows organisms to avoid harsh environment and to increase the possibility of survival. To investigate the molecular link between circadian and hibernating rhythms in the greater tube-nosed bats, Murina leucogaster, we set out to identify circadian genes that are expressed in bats, with specific focus on the PAS domain by using PCR-based screens. We could isolate a eDNA clone, designated as LPAS1, that encodes a protein of 521 amino acid residues. LPAS1 is closely related with CLOCK family with the highest homology to human CLOCK. Based on RT-PCR analyses, LPAS1 transcripts are ubiquitously present in tissues from both summer active and winter dormant periods. Given that LPAS1 is a member of the bHLH-PAS protein superfamily but lacks polyglutamine transactivation domains, it is likely to function as a repressor for endogenous CLOCK to hinder its roles in promoting transcription. Our result will open a new avenue to further examine the functional interconnection between the circadian clock and the circannual clock such as mammalian hibernation.

• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.82-96
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• 1991

To understand the mechanism of illegitimate recombination in mammalian cells, we have examined the recombination role of DNA topoisomerase II (Topo II ). We found that purified calf thymus Topo II mediates recombination between two phage $\lambda$ DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus Topo II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletion, and most crossovers take place between nonhomologous sequences of $\lambda$ DNA, as judged by the sequences of recombination junctions. In order to study the effects of Topo II on illegitimate recombination in mammalian cells, we have developed a new shuttle vector, pNKl, which contains three bacterial genes, amp(APR), galK and neo($Km^R$). Using this system, we have shown that a Topo II inhibitor, VM26, stimulated deletion formation in pNK1 DNA in monkey COS1 cells. Both in vitro and in vivo results suggest that Topo II participates in illegitimate recombination in mammalian cells.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.231-234
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• 1991

In the present investigation we have studied the effect of bear's gall on mammalian cells and demonstrated that COS-7 cells, which were derived Monkey kidney cells, had shown almost same extent of growth with 78 hrs in 10% FCS Dulbecco's Modified Eagle's medium with bear's gall and without bear's gall. But the hybridoma cells which were fused murine myeloma cells and the rat spleen cells for monoclonal antibody production died almost within 48 hrs. To investigate the effect of biosynthetic mechanism, cDNA were transfected to COS-7 cells, and it was shown that cDNA-transfected COS-7 cell had produced 30-40% less the amount of recombinant protein than the medium without bear's gall.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.155-162
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• 2004

In mammalian, cytochrome P4501A1 (CYP1A1) is very important for metabolism of xenobiotics such as PAHs(Polycyclic aromatic hydrocarbon) and heterocyclic amine, and it is induced by environmental contaminants such as PAHs, TCDD(2,3,7,8-tetrchlorodibenzo-p-dioxin) and 3-MC (3-methylcholanthrene). In fish, like mammalian, when it is exposed to environmental contaminants, they cause specific and sensitive induction of CYP1A. Therefore, induction of CYP1A in fish and mammalian is widely used as a biomarker for exposure of environmental contaminants. In this study, to compare the function of Cyp1a1 in fish with it in mammalian, we have used rainbow trout(Oncorhynchys mykiss) hepatoma cells (RTH-149) and mouse hepatocyte (Hepa-I). in order to examine induction of Cyp1a1 by TCDD, we have used the bioassay system. We examined effects of TCDD on the Cyp1a1-luciferase reporter gene activity, 7-ethoxyresorufin O-deethylase(EROD) activity and Cypa mRNA level.

• Molecules and Cells
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• v.25 no.1
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• pp.91-98
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• 2008

The Glu/$Asp^{7.32}$ residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with $Arg^8$ of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/$Asp^{7.32}$ also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking $Glu^{7.32}$ are important for antagonist as well as agonist selectivity.

• Toxicological Research
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• v.27 no.1
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• pp.25-29
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• 2011

The cytochrome P450 (P450, CYP) are the superfamily of heme-containing monooxygenase enzymes, found throughout all nature including mammals, plants, and microorganisms. Mammalian P450 enzymes are involved in oxidative metabolism of a wide range of endo- and exogenous chemicals. Especially P450s involved in drug metabolisms are important for drug efficacy and polymorphisms of P450s in individuals reflect differences of drug responses between people. To study the functional differences of CYP2A6, CYP2D6, and CYP4A11 variants, we cloned the four CYP2A6, three CYP2D6, and three CYP4A11 variants, which were found in Korean populations, in mammalian expression vector pcDNA by PCR and examined their expressions in COS-7 mammalian cells using immunoblots using P450 specific polyclonal antibodies. Three of four CYP2A6, two of three CYP4A11, and two of three CYP2D6 variants showed expressions in COS-7 cells but the relative levels of expressions are remarkably different in those of each variants. Our findings may help to study and explain the differences between functions of CYP variants and drug responses in Korean populations.