OBJECTIVETo investigate the roles of mouse erythroid differentiation and denucleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation.

METHODSMouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcDNA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate, mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and beta-globin genes were analysed by semi-quantitative RT-PCR.

RESULTSMEL cells transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index, and colony-forming rate in semi-solid medium (P<0.01). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of beta-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3.1), and the expression of c-myc decreased by 66.3%.

CONCLUSIONSMEDDF can induce differentiation of MEL cell and suppress its malignancy.

Activins ; genetics ; pharmacology ; Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Friend murine leukemia virus ; Globins ; biosynthesis ; genetics ; Inhibin-beta Subunits ; genetics ; pharmacology ; Leukemia, Erythroblastic, Acute ; metabolism ; pathology ; Mice ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Transfection ; Tumor Cells, Cultured

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

OBJECTIVES: To evaluate the effect of glutathione(GSH) on lead induced modulation of nitric oxide(NO) synthesis, and to examine how lead modulates NO production in macrophages. METHODS: This study was observed in a culture of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that was induced by the Abelson murine leukemia virus. The compounds investigated were lead chloride, N-acetyl-cystein(NAC), and Buthionine Sulfoximine(BSO). RESUJLTS: ATP synthesis in RAW 264.7 cells was unchanged by each lead concentration exposure in a dose dependent manner. The NO synthesis was decreased when exposed to lead(PbCl2) concentration 0.5 micro M. The presence of 300 micro M NAC, used as a pretreatment in the culture medium, caused the recovery of the lead induced decrease in NO synthesis, but in the presence of 300 micro M BSO as a pretreatment, there was no recoverey. Pretreatment with NAC and BSO had no affect on ATP synthesis at any of the lead concentrations used. CONCLUSIONS: These results indicated that GSH has a protective effect toward lead toxicity, and suggested that the inhibition of NO production in macrophage due to lead toxicity may be related to cofactors of iNOS (inducible nitric oxide synthase)
Abelson murine leukemia virus ; Acetylcysteine ; Adenosine Triphosphate ; Animals ; Buthionine Sulfoximine ; Glutathione* ; Macrophages ; Mice ; Nitric Oxide

The Bcr-Abl oncogene is produced by the reciprocal translocation between c-Abl gene on chromosome 9 and the Bcr gene on chromosome 22 in human genome. The encoded Bcr-Abl fusion protein is responsible for the pathogenesis of certain human leukemias. Abelson murine leukemia virus (A-MuLV) is a retrovirus that could lead to transformation of B lymphocyte in mice, and v-Abl is the oncogene of A-MuLV. Abl oncoproteins (such as Bcr-Abl and v-Abl) play critical roles in tumorigenesis of certain cell types. Several signal transduction pathways, including JAK/STAT/Pim, PI3K/AKT/mTOR and RAS/RAF/MEK signaling pathway, are involved in Abl-mediated tumorigenesis. In addition, Abl-mediated tumorigenesis is associated with mutation or abnormal modification of key signal molecules as well as dysregulation of some critical long noncoding RNAs (lncRNAs). Here, we review the molecular mechanisms by which Abl oncogenes activate three major signaling pathways, and provide a scientific basis for therapy of Abl oncoprotein-induced tumors.
Abelson murine leukemia virus ; Animals ; Cell Transformation, Neoplastic ; Fusion Proteins, bcr-abl ; Genes, abl ; Humans ; Phosphatidylinositol 3-Kinases

The genomic diversity of Avian leukosis virus subgroup J (ALV-J) was investigated in an experimentally infected chicken. ALV-J variants in tissues from four different organs of the same bird were re-isolated in DF-1 cells, and their gp85 gene was amplified and cloned. Ten clones from each organ were sequenced and compared with the original inoculum strain, NX0101. The minimum homology of each organ ranged from 96.7 to 97.6%, and the lowest homology between organs was only 94.9%, which was much lower than the 99.1% homology of inoculum NX0101, indicating high diversity of ALV-J, even within the same bird. The gp85 mutations from the left kidney, which contained tumors, and the right kidney, which was tumor-free, had higher non-synonymous to synonymous mutation ratios than those in the tumor-bearing liver and lungs. Additionally, the mutational sites of gp85 gene in the kidney were similar, and they differed from those in the liver and lung, implying that organ- or tissue-specific selective pressure had a greater influence on the evolution of ALV-J diversity. These results suggest that more ALV-J clones from different organs and tissues should be sequenced and compared to better understand viral evolution and molecular epidemiology in the field.
Animals ; Avian Leukosis Virus* ; Avian Leukosis* ; Birds ; Chickens* ; Clone Cells ; Kidney ; Liver ; Lung ; Molecular Epidemiology ; Silent Mutation

OBJECTIVES: Nickel (Ni) is present in many industrial working environments and consumer products, and is one of the leading cause of allergic contact dermatitis, which is a typical delayed (type IV) hypersensitivity reaction. However, the mechanism by which nickel causes this pathology is not well known. The contact dermatitis induced by nickel is mediated, primarily, through macrophages. This property was similar to autotoxicity related nitric oxide (NO) production. NO mediated cytotoxicity was dependent on both H2O2 and peroxynitrite (OONO-). The purpose of this study was to elucidate the role of NO/H2O2 in the cytotoxicity induced by nickel. Therefore, this study was designed to examine whether nickel could modulate NO/H2O2 production and how the Ni may affect ATP production, intracellular GSH level, and cell viability. METHODS: This study was based on the observations of cultures of RAW 264.7 cells, which originated from a tumor in a Balb/c mouse that had been induced by the Abelson murine leukemia virus. RAW 264.7 cells were treated with either Ni, N- onomethyl-L- arginine (NMLA), catalase, and DTT for 24-72 h. The cytotoxicity of the nickel was measured via the cell viability and NO2-, H2O2, GSH, and the mitochondrial function was evaluated by the adenosine triphosphate (ATP) production in the RAW 264.7 cells. RESULTS: The NO2- synthesis of RAW 264.7 cells increased with the increase in concentrations of Ni up to 50-micrometer, after 24 and 48 h of exposure, but then decreased at concentrations greater than 50-micrometer, and with time periods exceeding 48 h. In contrast, viability of cells and intracellular GSH level decreased in the presence of Ni in a dose and time dependent manner. However, the H2O2 synthesis of RAW 264.7 cells was not changed in the all experimental conditions. The NO2- synthesis of the cells was higher than control, whereas ATP, GSH and viability were lower than control in addition of Ni and the pretreatment of catalase or DTT prior to addition of Ni. CONCLUSIONS: These results suggest that NO plays an important role in the cytotoxicity of Ni. Cytotoxicity of Ni may exert through modulation of NO production and associate with a decrease in intracellular GSH levels.
Abelson murine leukemia virus ; Adenosine Triphosphate ; Animals ; Arginine ; Catalase ; Cell Survival ; Dermatitis, Allergic Contact ; Dermatitis, Contact ; Hydrogen Peroxide* ; Hydrogen* ; Hypersensitivity ; Macrophages ; Mice ; Nickel* ; Nitric Oxide* ; Pathology ; Peroxynitrous Acid

OBJECTIVES: The nickel and cobalt present in many industrial working environments and consumer products. They are two of the leading causes of allergic contact dermatitis, which is a typical delayed(type IV) hypersensitivity reaction. However, the mechanism by which nickel and cobalt causes this pathology is not well known. The nickel and cobalt induced contact dermatitis is mediated primarily through macrophages. This mechanism is similar to the autotoxicity procedure for NO. Therefore, this study was designed to examine whether the metals could modulate NO production and how the metals may affect ATP production and cell viability. In summary, the purpose of this study was to elucidate the role of NO in the nickel and cobalt induced cytotoxicity. METHODS: This study is based on observations of cultures of RAW 264.7 cells which are originated from a tumor of Balb/c mouse that was induced by Abelson murine leukemia virus. RAW 264.7 cells were treated with either Ni, Co, Ni plus Co, or Nmonomethyl-L- arginine(NMLA) for 24-72 h. The cytotoxicity of the nickel and cobalt was measured by cell viability and NO2-, and mitochondrial function was evaluated by adenosine triphosphate(ATP) production in RAW 264.7 cells. In addition, the morphology of cells was observed using an inverted microsope. RESULTS: The NO2- synthesis of RAW 264.7 cells increased with increasing concentrations of Ni and Co up to 50 microM after 24 and 48 h of exposure to Ni and Co but then decreased if the concentration was greater than 50 microM and the time period was greater than 48 h. However, the viability of cells was decreased by Ni and Co exposure in a dose and time dependent manner. Therefore, 50 microM Ni or Co and 48 h of treatment were used in this study. A complete inhibition of NO2- synthesis by Ni or/and Co occurred when iNOS inhibitor, NMLA, were pretreated prior to addition of Ni or/and Co, whereas Ni or/and Co induced decrease of synthesis of ATP and viability completely recovered when NMLA were pretreated prior to addition of Ni or/and Co. Ni or/and Co(50 microM) induced the characteristic morphological features of cytotoxicity which is characterized by a shrinkage of cytoplasm and irregular shape of the cells, but the pretreatment of NMLA resulted in a recovered morphological change of the cells to their normal appearance. CONCLUSIONS: These results suggest that NO plays an important role in the pathogenesis of the cytotoxicity of nickel and cobalt, and nickel and cobalt may exert their toxicities by means of modulation of NO production. The results from this study may facilitate further understanding the role of NO on nickel and cobalt induced immune and inflammatory processes.
Abelson murine leukemia virus ; Adenosine ; Adenosine Triphosphate ; Animals ; Cell Survival ; Cobalt* ; Cytoplasm ; Dermatitis, Allergic Contact ; Dermatitis, Contact ; Hypersensitivity ; Macrophages ; Metals ; Mice ; Nickel* ; Nitric Oxide* ; Pathology

In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; metabolism ; Breeding ; Chickens ; genetics ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Viral Envelope Proteins ; genetics ; metabolism

The transmembrane protein (TM) encoded by gp37 gene plays a critical role when virus fusion with cell membrane occurs. Several highly conserved regions in TM are important targets for antivirus studies. Studies on structure and function of TM will provide basic information for anti-retrovirus, especially for avian leukosis virus. In the study, gp37 gene was amplified by PCR from the Chinese strain ALV-J-WS0701. The gp37 gene was cloned into pMD18-T vector, and was sequenced. Then, pFast-BacHTb-gp37 vector was constructed and expressed by baculovirus expression vector system. The expression product of gp37 gene was analyzed by indirect immunofluorescence assay and Western blot. The results showed that positive green fluorescence was present in sf9 cells infected with recombinant virus and a protein band with a molecular weight of 21kD was present in Western blot. It is concluded that gp37 gene was expressed in sf9 cells infected with recombinant virus successfully.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; Cell Line ; Chickens ; Cloning, Molecular ; Gene Expression ; Spodoptera ; Viral Envelope Proteins ; genetics ; metabolism

To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P < 0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; immunology ; pathogenicity ; Chick Embryo ; Fibroblasts ; virology ; Proliferating Cell Nuclear Antigen ; analysis ; immunology ; Viral Load ; immunology