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ABSTRACT: The fission yeast Schizosaccharomyces pombe is a useful model organism for studying a variety of eukaryotic cellular events such as the cell cycle control mechanisms. For inducible expression of exogenous genes in S. pombe, vectors carrying the nmt1 (no message in thiamine 1) promoter are most commonly used. Although nmt1 is a potent promoter, its transcription activity is drastically repressed in the presence of a low concentration of thiamine. Therefore, a combination of thiamine and nmt1 promoter is convenient for regulating gene expression in an all-or-none fashion. However, it has been difficult to adjust the nmt1 promoter activity in a controlled manner. Here we describe a chemical compound, designated as YAM2, whose repressive activity on the nmt1 promoter has a wider linear range than thiamine. Expression of exogenous proteins, such as human immunodeficiency virus type 1 Vpr and jellyfish green fluorescent protein, driven by the nmt1 promoter is gradually repressed by YAM2 in a dose-dependent manner. YAM2 does not exhibit a detectable level of cytotoxicity at a concentration required to fully repress the nmt1 promoter. The compound may serve as a useful tool for controlled expression of the nmt1-driven gene in S. pombe.
Analytical Biochemistry 02/2011; 412(2):159-64. · 3.00 Impact Factor
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The Lancet Infectious Diseases 06/2009; 9(5):266-7. · 17.39 Impact Factor
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ABSTRACT: Lentiviral accessory proteins are thought to play important roles in regulating the viral replication through modulation of host cell functions. For example, Vpr of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G2 arrest in a host cell-specific manner. Similarly, HIV-2 Vpr, but not Vpx, has been shown to induce G2 arrest in primate cells. It has also been reported that Orf-A of feline immunodeficiency virus (FIV) induces G2 arrest in a simian cell line. However, activities of these non-HIV-1 accessory proteins in different cellular context are unclear. In this study, effects of HIV-2 Vpr, Vpx and FIV Orf-A on cell cycle progression were compared with those of HIV-1 Vpr in various mammalian cell lines and the fission yeast. These non-HIV-1 accessory proteins induced the cell cycle arrest in a host cell-specific manner, and their specificities were different from each other. Interestingly, HIV-2 Vpx-induced G2 arrest in bovine MDBK cells. It was also notable that HIV-2 Vpx and FIV Orf-A appeared to block the cell separation in the fission yeast. The host cell-specific activities of different lentiviral accessory proteins revealed in this study may provide a useful basis for elucidating the mechanism of their functions.
Microbes and Infection 05/2009; 11(6-7):646-53. · 3.10 Impact Factor
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ABSTRACT: Mouse cationic amino acid transporter 1 (mCAT1) serves as the receptor for ecotropic murine leukemia virus (eMuLV). It has been shown that mCAT1 is expressed on the basolateral surface of polarized epithelial MDCK cells. However, little is known about the mechanisms involved in the intracellular trafficking of mCAT1. Using the green fluorescent protein-tagged mCAT1 expressed in MDCK cells, we report here that mCAT1 is physically associated with clathrin adaptor protein complex 1 (AP-1) implicated in protein trafficking from trans-Golgi network (TGN) to the basolateral surface. When the cells were infected with eMuLV, reduction of cell surface mCAT1, as well as a concomitant decrease in mCAT1-AP-1 association, was observed while association of mCAT1 with AP-3 involved in the TGN-to-lysosome trafficking was increased. Similar results were obtained when eMuLV envelope protein alone was expressed. The results may provide useful insights into the mechanism by which a simple retrovirus downregulates its receptor.
Virology 12/2007; 368(2):342-50. · 3.35 Impact Factor
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ABSTRACT: Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.
Microbes and Infection 11/2006; 8(12-13):2736-44. · 3.10 Impact Factor
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Michiaki Masuda
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ABSTRACT: Human immunodeficiency virus (HIV) is a causative agent of acquired immunodeficiency syndrome (AIDS) and a member of Retrovirus family. The name of "retrovirus" is said to be derived from "reverse-transcribing oncogenic virus." Living up to its name, retrovirus has contributed to oncology, especially in the field of cancer pathogenesis. Retrovirus research has led to discovery of a number of oncogenes as well. Since the discovery of HIV in 1983, however, retrovirus has also been considered as an important etiologic agent that could incapacitate the cells involved in immune responses. As of the end of 2004, the number of people living with HIV/AIDS is estimated to be as large as 40 million. Every year, nearly 5 million people are newly infected with HIV, and 3 million people die of AIDS in the world, mainly in Africa and southeastern and southern Asia. Despite extensive studies, detailed mechanisms of HIV pathogenesis are still unclear, and efforts are being made to clarify functions of various HIV proteins and identify the cellular factors that could interact with the HIV proteins. One of the HIV accessory proteins, Vpr, causes the host cell cycle arrest at G2 phase, which may play an important pathogenic role in AIDS induction. Exploiting the fission yeast Schizosaccharomyces pombe useful for cell cycle studies, I've been trying to elucidate the mechanism by which Vpr induces the G2 arrest as presented in this review.
Rinsho byori. The Japanese journal of clinical pathology 11/2005; 53(10):950-6.
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ABSTRACT: S-phase kinase-associated protein 2 (SKP2), a member of the F-box family of ubiquitin-protein ligase complexes, controls the stability of cell cycle-related proteins including p27Kip1. The authors examined how the expression level of SKP2 affects the expression level of cell cycle-related proteins, cell cycle status, viability, and chemoresistance in A549 lung adenocarcinoma cells. Overexpression of SKP2 reduced the expression of p27Kip1, cyclin E, and p21Cip1, increased S-phase cells, rescued A549 cells from apoptosis due to adenoviral infection, and also increased chemoresistance against camptothecin, cisplatin, and AG1478. Down-regulation of SKP2 did not affect cell cycle status, and reduced cell viability.
Experimental Lung Research 01/2005; 30(8):687-703. · 1.22 Impact Factor
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ABSTRACT: We investigated how nutritional deficiency affects cell cycle and cell viability in A549 lung adenocarcinoma cells. Deprivation of various amino acids or glucose induced cell cycle arrest and cell death in a different manner. Cell death on deprivation of these nutrients was increased by downregulating of p27Kip1 with RNA interference. It was also observed that intrinsic p27Kip1 was segregated in cytoplasm in a glucose-deprived situation. In conclusion, amino acid or glucose deprivation induced cell cycle arrest and cell death, part of which is thought to be rescued by the existence of cytoplasmic p27Kip1.
Cancer Letters 10/2004; 213(1):99-109. · 4.24 Impact Factor
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ABSTRACT: Glutathione S-transferase P1 (GSTP1) belongs to xenobiotic enzymes, and is supposed to contribute to chemoresistance. Though it was reported that GSTP1 gene is suppressed by cytosine-guanine (CpG) island methylation of its promoter, this promoter is not strongly methylated and GSTP1 protein is highly expressed in lung cancer. We intended to induce methylation of GSTP1 CpG island by using a methylated sense oligonucleotide complementary to this region. When we transduced the methylated oligonucleotides to A549 lung adenocarcinoma cells, methylation of the GSTP1 promoter and reduction of GSTP1 expression was induced, cell viability was reduced; however, chemoresistance against cisplatin has not clearly changed.
Cancer Letters 09/2004; 212(2):211-23. · 4.24 Impact Factor
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ABSTRACT: Glutathione S-transferase P1 (GSTP1) is one of the important xenobiotic-metabolizing enzymes. It was reported that GSTP1 was overexpressed in malignant tissues, and its expression level was associated with resistance to chemotherapeutics. We carried out transfection of GSTP1 sense and antisense vectors to examine effects of GSTP1 on cell cycle arrest and apoptosis induced by camptothecin in HeLa cells. Transfection of GSTP1 antisense vector induced apoptosis. Camptothecin-induced S- or G2/M arrest was intensified by transfection of GSTP1 antisense vector, and subsequent apoptosis was attenuated by transfection of GSTP1 sense vector. These results suggest that GSTP1 has protective effects against camptothecin-induced cytotoxicity.
Cancer Letters 02/2004; 203(2):199-207. · 4.24 Impact Factor
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ABSTRACT: An accessory protein, Vpr, of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G(2)/M arrest in primate cells, but not in rodent cells, suggesting that a species-specific factor might be involved in the phenomenon. To study whether Vpr can cause G(2)/M arrest in non-primate cells, a novel adenoviral vector, Ad-VIG, co-expressing HIV-1 Vpr and green fluorescent protein (GFP) was constructed and infected on cell lines derived from various mammalian species. With its ability to express GFP, Ad-VIG enabled flow cytometric evaluation of transduction efficiency in the infected cells, and Western blot analysis showed successful expression of Vpr in the vector-transduced cells. Upon Ad-VIG infection, human HeLa, African green monkey Vero, feline CRFK, and bovine MDBK cells manifested cell cycle G(2)/M arrest. This is the first study showing that non-primate feline and bovine cells are susceptible to Vpr-induced cell cycle arrest.
Biochemical and Biophysical Research Communications 12/2003; 311(3):748-53. · 2.48 Impact Factor
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ABSTRACT: PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) which causes a rapidly progressive spongiform neurodegenerative disease in rodents. The primary target of PVC-211 MuLV infection in the brain is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection. Previous studies have shown that changes in the envelope gene of PVC-211 MuLV confer BCEC tropism to the virus. However, little is known about how infection of BCECs by PVC-211 MuLV induces neurological disease. Previous results suggest that nitric oxide (NO), which has been implicated as a potential neurotoxin, is involved in PVC-211 MuLV-induced neurodegeneration. In this study, we show that expression of inducible nitric oxide synthase (iNOS), which produces NO from L-arginine, is induced in BCECs from PVC-211 MuLV-infected rats. Furthermore, elevated levels of a 32-kDa cellular protein modified by 3-nitrotyrosine, which is a hallmark of NO production, were observed in virus-infected BCECs. BCECs from rats infected with BCEC-tropic but nonneuropathogenic PVF-e5 MuLV, which is a chimeric virus between PVC-211 MuLV and F-MuLV, fail to induce either iNOS expression or elevation of tyrosine nitration of a 32-kDa protein. These results suggest that expression of iNOS and nitration of tyrosine residues of a 32-kDa protein in PVC-211 MuLV-infected BCECs may play an important role in neurological disease induction.
Journal of Virology 06/2003; 77(9):5145-51. · 5.40 Impact Factor
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ABSTRACT: Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.
Journal of Virology 07/2002; 76(11):5540-7. · 5.40 Impact Factor
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ABSTRACT: Novel sets of env gene PCR primers for distinguishing human immunodeficiency virus type 1 (HIV-1) subtypes B and E were designed. These primers anneal to different regions of the env gene and amplify DNA fragments of distinct sizes in a subtype-specific manner. Blood samples from 11 HIV-1 carriers in Thailand and 46 carriers in Japan were examined by PCR. The new env primers detected HIV-1 proviral DNA in 100% (11/11) and 88% (37/42) of the subtype B and E infection cases, respectively. The env primers also detected proviral DNA in saliva and breast milk samples in seven of 11 cases and two of three cases, respectively. The PCR subtyping results matched completely with those obtained by nucleotide sequencing of the env V3 region. The results suggest that the PCR using the env primers designed in this study may be an accurate and cost-effective method for differentiating subtypes B and E of HIV-1 in a large number of clinical samples. However, subtype E specific primer cross-react with subtype A, C, G, the new primer in this study is useful for regions in South East Asia where subtype E is predominant.
Journal of Virological Methods 04/2002; 101(1-2):11-20. · 2.01 Impact Factor
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ABSTRACT: Glutathione S-transferase P1 (GSTP1) is one of the xenobiotic-metabolizing and antioxidant enzymes, identified in the peripheral lungs. Recently, the authors reported the association between GSTP1 gene polymorphism and susceptibility to chronic obstructive pulmonary disease (COPD), and protective effect of GSTP1 against cigarette smoke in human lung fibroblasts in vitro. In this study, the authors investigated that depletion of GSTP1 by itself could induce cell death, including apoptosis, in human lung fibroblast-derived HFL-1 cells. The level of apoptosis and necrosis was increased significantly with GSTP1 antisense vector transfection. It was also observed that the transfection efficiency and the expression level of the vector were weaker in the transfectant of the antisense vector than in those of the sense and control vectors, which is also thought to indicate that inhibition of GSTP1 expression by the antisense vector alone affects cellular viability. However, there was no difference among these transfectants neither on glutathione (GSH) level nor on c-Jun NH2-terminal kinase (JNK) activation. Therefore, the authors report here that underexpression of GSTP1 appeared to induce apoptosis on lung fibroblasts, which suggests that GSTP1 may have protective effects against apoptosis in the airway cells, though the mechanism of this apoptotic pathway is still to be elucidated.
Experimental Lung Research 29(7):523-36. · 1.22 Impact Factor
