The Rana grylio virus (RGV) is a member of the genus Ranavirus. It belongs to the family Iridoviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed Δ67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluorescence gene (EGFP) as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosum cyprini (EPC) cells were infected with the resulting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally Δ67R-RGV was generated. Total DNA of Δ67R-RGV infected cells was extracted for PCR analyses. Simulateously, mock infected and wild-type RGV (wt-RGV) infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in Δ67R-RGV infected cells. Furthermore, one-step growth curves of wt-RGV and Δ67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essential for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.
Genes, Viral ; physiology ; Genome, Viral ; Polymerase Chain Reaction ; Pyrophosphatases ; genetics ; Ranavirus ; genetics ; Recombination, Genetic

As an important drug during maintenance treatment of acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) has several side effects, including hepatotoxicity and bone marrow suppression. Since its tolerability varies from person to person, 6-MP treatment should be individualized. The deficiency of thiopurine methyltransferase (TPMT) enzyme activity is associated with 6-MP intolerance. There is a lower frequency of mutation in TPMT alleles among Asian patients. Recent studies have shown that in ALL patients with NUDT15 gene mutation, the maximum tolerated dose of 6-MP is lower than the conventional dose. The article reviews the significance of NUDT15 gene in individualized treatment with 6-MP in children with ALL.
Antimetabolites, Antineoplastic ; Child ; Humans ; Mercaptopurine ; Methyltransferases ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Pyrophosphatases ; genetics

Anemia ; chemically induced ; Hepatitis C, Chronic ; drug therapy ; genetics ; Humans ; Polymorphism, Genetic ; Pyrophosphatases ; genetics ; Ribavirin ; adverse effects ; therapeutic use

OBJECTIVETo study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2.

METHODSRecombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells.

RESULTSPC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells.

CONCLUSIONRNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.

Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Pyrophosphatases ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; Transfection

OBJECTIVE: To investigate the association between single nucleotide polymorphism of NUDT15 gene (SNP rs116855232) and hepatotoxicity in children with acute lymphocytic leukemia (ALL). METHODS: A total of 135 children with ALL in Shandong Province were recruited in this study, and patients were divided into two groups based on the presence of liver injury. Genotypes of each patient were detected using PCR and Sanger sequencing. Clinical data and the average dose of 6-mercaptopurine (6-MP) were collected and analyzed by SPSS 19.0 software. RESULTS: Respectively, 99 patients were found with CC genotype, 32 patients with CT genotype and 4 patients with TT genotype. Compared with ALL patients without hepatotoxicity, there was a difference in genotypes between the two groups in the initial stage of chemotherapy for leukemia (Chi CONCLUSION The polymorphism of rs116855232 in NUDT15 gene was associated with hepatotoxicity induced by 6-mercaptopurine in children with ALL, and ALL patients with TT genotype should take a lower dose of 6-MP to avoided hepatotoxicity.
Antimetabolites, Antineoplastic/therapeutic use* ; Chemical and Drug Induced Liver Injury/genetics* ; Child ; Genotype ; Humans ; Mercaptopurine/adverse effects* ; Polymorphism, Single Nucleotide ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Pyrophosphatases/genetics*

Mercaptopurine is a common chemotherapeutic drug and immunosuppressive agent and plays an important role in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. It may cause severe adverse effects such as myelosuppression, which may result in the interruption of treatment or complications including infection or even threaten patients' lives. However, the adverse effects of mercaptopurine show significant racial and individual differences, which reveal the important role of genetic diversity. Recent research advances in pharmacogenomics have gradually revealed the genetic nature of such differences. This article reviews the recent research advances in the pharmacogenomics and individualized application of mercaptopurine.
Antimetabolites, Antineoplastic ; therapeutic use ; Humans ; Mercaptopurine ; metabolism ; therapeutic use ; Methyltransferases ; genetics ; Pharmacogenetics ; Polymorphism, Genetic ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; Pyrophosphatases ; genetics

OBJECTIVEFindings from the previous studies have suggested a relationship between ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP-1) or plasma cell membrane glycoprotein 1 (PC-1) gene single nucleotide polymorphism (K121Q, rs1044498) and genetic susceptibility to obesity. However, such relationship is not reproduced by some currently available studies. In this context, the present study is aimed to quantitatively analyze the association of K121Q variant with obesity in all published case-control studies in European adult populations.

METHODSPublished literature from PubMed, EMBASE, and ISI web of science databases were retrieved. The studies evaluating the association of ENPP1/PC1 gene K121Q polymorphism with obesity were included, in which sufficient data were presented to calculate the odds ratio (OR) with 95% confidence intervals (CIs).

RESULTSTen case-control studies meeting the inclusion criteria identified a total of 24,324 subjects including 11,372 obese and 12,952 control subjects. The meta-analysis results showed a statistically significant association of K121Q with obesity [OR (95%CI): 1.25 (1.04-1.52) P=0.021] under a recessive model of inheritance (QQ vs. KK+KQ) without heterogeneity or publication bias.

CONCLUSIONSThe results from the present study have indicated that ENPP1/PC1 Q121 variant may increase the risk of obesity and that more well-designed studies based on a larger population will be required to further evaluate the role of ENPP1/PC1 gene K121Q polymorphism in obesity and other related metabolic syndromes.

Europe ; epidemiology ; Genetic Predisposition to Disease ; Humans ; Obesity ; epidemiology ; genetics ; Odds Ratio ; Phosphoric Diester Hydrolases ; genetics ; Polymorphism, Genetic ; Pyrophosphatases ; genetics ; Risk Factors

Type 2 diabetes is characterized by insulin resistance, and ENPP1 plays an important role in insulin resistance. We investigated the association of the ENPP1 K121Q polymorphism with both diabetes and obesity (body mass index [BMI]) in Korean male workers. The study design was case-control. Subjects were 1,945 male workers (type 2 diabetes, 195; non-diabetes, 1,750) of nuclear power plants who received examinations from March to October in 2004. We collected venous blood samples under fasting (> or =8 hr) conditions, calculated BMI by height and weight, and assessed relevant biochemical factors. The results of this study demonstrated that the ENPP1 121Q genotype (KQ+QQ types) was not associated with type 2 diabetes (odds ratios [OR], 0.854; 95% confidence interval [CI], 0.571-1.278) or obesity (OR, 0.933; 95% CI, 0.731-1.190). In addition, the frequency of the Q allele was not related to type 2 diabetes (OR, 0.911; 95% CI, 0.630-1.319) or obesity (OR, 0.962; 95% CI, 0.767-1.205). We concluded that the ENPP1 121Q allele is not a critical determinant for either diabetes or obesity in Korean males. The discordance between the results of this study and those derived from studies of Dominican, South Asian, Caucasian, Finnish, and French populations might be due to differences in genetic backgrounds between these populations.
Adult ; Diabetes Mellitus, Type 2/*ethnology/*genetics ; Employment ; Gene Frequency ; Genetic Predisposition to Disease/ethnology ; Genotype ; Humans ; Korea/epidemiology ; Male ; Middle Aged ; Obesity/*ethnology/*genetics ; Phosphoric Diester Hydrolases/*genetics ; Polymorphism, Genetic ; Prevalence ; Pyrophosphatases/*genetics

BACKGROUNDEctonucleotide pyrophosphatase/phosphodiesterase (ENPP)-1 is a membrane-bound protein that catalyzes the hydrolysis of extracellular nucleoside triphosphates to monophosphate and extracellular inorganic pyrophosphate (ePPi). Mechanical stimulation regulates ENPP-1 expression. This study sought to investigate the changes in ENPP-1 expression after stimulation using cyclic mechanical tension (CMT).

METHODSRat end-plate chondrocytes were cultured and subjected to CMT (at 3%, 6%, and 9% elongation) for 20, 40, and 60 minutes to observe changes in the expression of ENPP-1. To investigate the pathway, end-plate chondrocytes were exposed to 10 ng/ml of transforming growth factor beta 1 (TGF-β1), TGF-β1 siRNA, or a specific extracellular signalregulated kinase (ERK)1/2 inhibitor, U0126, in addition to CMT. Changes in ENPP-1 expression were measured by reverse transcription PCR (RT-PCR) and Western blotting.

RESULTSWe observed the largest increase in ENPP-1 expression following 3% elongation CMT stimulation. ENPP-1 expression was also increased when end-plate chondrocytes were exposed to 10 ng/ml of TGF-β1, but decreased after TGF-β knockdown with siRNA. ERK1/2 phosphorylation was activated after 3% elongation for 40 minutes, and the stimulatory effect of TGF-β1 on ENPP-1 mRNA and protein expression was inhibited by the suppression of the ERK1/2 pathway using U0126.

CONCLUSIONCMT increases the expression of ENPP-1 in end-plate chondrocytes in a manner likely dependent on TGF-β induction by the ERK1/2 signaling pathway.

Animals ; Blotting, Western ; Cells, Cultured ; Chondrocytes ; metabolism ; Phosphoric Diester Hydrolases ; genetics ; metabolism ; Pyrophosphatases ; genetics ; metabolism ; RNA, Small Interfering ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Stress, Mechanical ; Transforming Growth Factor beta1 ; genetics ; metabolism

Metallothionein-3 (MT-3), renamed as growth inhibitory factor (GIF), is a brain specific member of the metallothionein family. Human dUTPase is a recently found protein in brain that can interact with hMT-3. They have the growth inhibitory activity on neuron cell by interaction. To study the affection of hMT-3 to dUTPase's eliminating the cellular toxicity caused by dUTP, the pSVHA-dUTPase and pFLag-hMT-3 genes have been transfected into HEK293 cells. In addition, the dUTPase and hMT-3 proteins were expressed in BL21 to study the role of hMT-3 on the hydrolyzation of dUTP by dUTPase. The results demonstrate that the cells co-transfected with dUTPase and hMT-3 genes have more strong resistibility to dUTP than the cells transfected only with dUTPase gene. And that the hMT-3 protein can accelerate the hydrolyzation of dUTP by dUTPase. All these indicate that hMT-3 can cooperate with dUTPase to protect better the 293 cells from dUTP. This research offered the theoretic elements for the application of hMT-3 and dUTPase in chemic cure.
Cell Line ; Deoxyuracil Nucleotides ; antagonists & inhibitors ; chemistry ; Nerve Tissue Proteins ; chemistry ; genetics ; pharmacology ; Neurons ; cytology ; drug effects ; Protein Interaction Domains and Motifs ; Pyrophosphatases ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection