BACKGROUND: ADP-ribosyl pyrophosphatases (ADPRase) has been known to catalyze the hydrolysis of ADP-ribose to ribose-5-phosphate and AMP. The role of ADPRase has been suggested to sanitize the cell by removing potentially toxic ADP-ribose. In this study, we examined the effect of nitric oxide on ADPRase activity in macrophages. METHODS: ADPRase activity was measured in NO-inducing J774 cells. For in vitro experiments, recombinant human ADPRase was prepared in bacteria. RESULTS: ADPRase activity was increased by the treatment of exogenous NO generating reagent, sodium nitroprusside (SNP), in J774 cells. The increased ADPRase activity was mediated by the post-translational modification, likely to cause cADP-ribosylation via nitrosylation of cysteine residue on the enzyme. The stimulation with endogeneous NO inducers, TNF-alpha/IFN-gamma, also increased ADPRase activity through NO synthesis. Futhermore, ADPRase activity may be mediated by the post-translational modification of ADPRase, ADP-ribosylation. CONCLUSION: These results indicate that NO synthesized by macrophage activation plays a critical role in the increase in ADPRase activity following ADP-ribose metabolism.
Adenosine Diphosphate Ribose* ; Bacteria ; Cysteine ; Humans ; Hydrolysis ; Macrophage Activation ; Macrophages ; Metabolism ; Nitric Oxide* ; Nitroprusside ; Protein Processing, Post-Translational ; Pyrophosphatases

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

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

No abstract available.
Basophils/cytology/*metabolism ; Flow Cytometry ; HLA-DR Antigens/metabolism ; Humans ; Interleukin-3 Receptor alpha Subunit/metabolism ; Leukocyte Count ; Phosphoric Diester Hydrolases/metabolism ; Pyrophosphatases/metabolism ; Receptors, CCR3/metabolism ; Urticaria/*diagnosis/immunology/metabolism

OBJECTIVE: To investigate the relationship between DNA degradeation and postmortem interval. METHODS: dUTP was transferred to 3'terminal of DNA by using terminal deoxynucleotidyl transferase (TDT), then the reminders of dUTP after experimental reaction, as indicator of quantity of DNA degradation, were detected. RESULTS: The reminders of dUTP were decreasing along with the postmortem interval. CONCLUSION Postmortem DNA degradation may be used in postmortem interval judgment.
Animals ; Cell Nucleus/metabolism* ; Clinical Enzyme Tests/methods* ; DNA/metabolism* ; DNA Nucleotidylexotransferase/metabolism* ; Kidney/metabolism* ; Liver/metabolism* ; Male ; Postmortem Changes ; Pyrophosphatases/metabolism* ; Rats ; Rats, Sprague-Dawley ; Spectrophotometry, Ultraviolet ; Spleen/metabolism* ; Time Factors

Cementum is critical for anchoring the insertion of periodontal ligament fibers to the tooth root. Several aspects of cementogenesis remain unclear, including differences between acellular cementum and cellular cementum, and between cementum and bone. Biomineralization is regulated by the ratio of inorganic phosphate (Pi) to mineral inhibitor pyrophosphate (PPi), where local Pi and PPi concentrations are controlled by phosphatases including tissue-nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). The focus of this study was to define the roles of these phosphatases in cementogenesis. TNAP was associated with earliest cementoblasts near forming acellular and cellular cementum. With loss of TNAP in the Alpl null mouse, acellular cementum was inhibited, while cellular cementum production increased, albeit as hypomineralized cementoid. In contrast, NPP1 was detected in cementoblasts after acellular cementum formation, and at low levels around cellular cementum. Loss of NPP1 in the Enpp1 null mouse increased acellular cementum, with little effect on cellular cementum. Developmental patterns were recapitulated in a mouse model for acellular cementum regeneration, with early TNAP expression and later NPP1 expression. In vitro, cementoblasts expressed Alpl gene/protein early, whereas Enpp1 gene/protein expression was significantly induced only under mineralization conditions. These patterns were confirmed in human teeth, including widespread TNAP, and NPP1 restricted to cementoblasts lining acellular cementum. These studies suggest that early TNAP expression creates a low PPi environment promoting acellular cementum initiation, while later NPP1 expression increases PPi, restricting acellular cementum apposition. Alterations in PPi have little effect on cellular cementum formation, though matrix mineralization is affected.
Alkaline Phosphatase ; metabolism ; Animals ; Cell Line, Transformed ; Dental Cementum ; cytology ; metabolism ; physiology ; Gene Expression Profiling ; Humans ; Mice ; Models, Animal ; Phosphoric Diester Hydrolases ; metabolism ; Pyrophosphatases ; metabolism ; Tooth Root ; metabolism ; physiology ; X-Ray Microtomography

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

OBJECTIVE: To explore the value of flow cytometry in anaphylactic shock diagnosis by CD63 expression being detected using flow cytometry to conform the activation of basophils. METHODS: Sixteen rats were randomly divided into two groups: control group and anaphylactic shock group. The model of anaphylactic shock rat with ovalbumin injection was established. CD63, CD45 and CD203c antibody combination, flow cytometry was employed to detected blood basophil CD63 expression. Immunofluorescence method was employed to observe the CD63 immunofluorescence staining in the rat lung tissue. RESULTS: (1) Pure basophils were obtained by CD45 and CD203c gating. (2) The percentages of basophils CD63 were (17.34 +/- 2.04)% and (1.52 +/- 0.35)% in the experimental and control group, respectively. The differences between two groups were statistically significant (P < 0.01). (3) Compared with the control group, the expression of CD63 in basophils increased in anaphylactic shock lung tissue. CONCLUSION The detection of CD63 by flow cytometry could be the supplement of vivo allergic reactions and have good clinical value.
Anaphylaxis/metabolism* ; Animals ; Basophil Degranulation Test/methods* ; Basophils/metabolism* ; Biomarkers/analysis* ; Disease Models, Animal ; Female ; Flow Cytometry ; Lung/pathology* ; Male ; Ovalbumin/administration & dosage* ; Phosphoric Diester Hydrolases/immunology* ; Pyrophosphatases/immunology* ; Random Allocation ; Rats ; Rats, Wistar ; Tetraspanin 30/metabolism*

OBJECTIVE: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. METHODS: After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. RESULTS: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. CONCLUSIONS Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
Escherichia coli ; genetics ; Glucosides ; chemistry ; Humans ; Protein Domains ; Protein Stability ; Pyrophosphatases ; chemistry ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; chemistry ; isolation & purification ; TRPM Cation Channels ; chemistry ; isolation & purification ; Thrombin ; metabolism

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m⁷GDP and m²²⁷GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m⁷GDP cap from mRNAs in the presence of Mg²(+) or Mn²(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.
Amino Acid Motifs ; Biocatalysis ; Cell Nucleus ; enzymology ; Consensus Sequence ; Cytoplasm ; enzymology ; metabolism ; Guanosine Diphosphate ; metabolism ; Histidine ; metabolism ; Humans ; Hydrolysis ; Luciferases ; genetics ; Magnesium ; metabolism ; Manganese ; metabolism ; Mutagenesis ; Mutation ; Pyrophosphatases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; RNA Caps ; chemistry ; metabolism ; pharmacology ; RNA, Small Nucleolar ; chemistry ; metabolism ; pharmacology