Nucleoside diphosphate kinase (Ndk) is ubiquitous and highly conserved multifunctional key enzyme in nucleotide metabolism. It generates nucleoside triphosphates (NTPs) by transfer of gamma-phosphates from nucleoside triphosphates such as ATP or GTP to nucleoside diphosphate. The formation of an autophosphorylated enzyme intermediate is involved in that mechanism. The phosphate is usually supplied by ATP and Ndk activity in different subcellular compartments. Ndk may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. Ndk is playing an important role in bacterial pathogenesis and emerging evidences recognize multiple roles of Ndk in host-microbe interaction. Here, I review some examples of the role of Ndk in intra- and extracellular microorganism.
Adenosine Triphosphate ; Guanosine Triphosphate ; Nucleoside-Diphosphate Kinase

BACKGROUND: Uracil nucleotides are stored in platelets and all other cells, and are released into the extracellular space upon stimulation. They show various biological responses but their actions and mechanism are not well understood. This study was conducted to investigate the effects of uridine 5'-triphosphate(UTP) on vascular tone and to identify the characteristics of their receptors. METHODS: Aortic ring preparation were made from the rat descending thoracic aorta. Endo-thelial cells were preserved or removed by gentle rubbing, The basal tension of aortic ring was lgm and isometric contraction were recorded on polygraph using force transducer. RESULTS: In aortic ring Precontracted by 100nM norepinephrine, UTP induced dual effect with various concentrations. UTP elicited endothelium-dependent relaxation at low concentrations(100nM-10microM), and endothelium-independent contraction at high concentrations(more than 30microM). Among uracil nucleotides, UDP was as much effective as UTP in vascular tone, but UMP and uridine were not. UTP(pA50 6.15) was more potent than ATP(5.17), ITP(4.75) and other nucleotides(TTP, GTP, CTP). At basal tension, UTP induced relaxation at low concentrations and contraction at hige concentrations in endothelium-intact ring. But in endothelium-removed ring, UTP elicited only contraction. Prior treatment of aortic ring with suramin, a non-selective P2-purinoceptor blocker, inhibited UTP-Induced relaxation and contraction. Reactive blue-2, a P2gamma purinoceptor blocker, inhibited relaxation only, but alpha, beta-methylene ATP, a P2x Purinoceptor blocker, enhanced contractile response. ATP inhibited the UPT-induced relaxation, but 2-methylthio ATP did not alter the effects of UTP. It means that UTP and ATP act at the same receptor but 2-methylthio ATP does not. CONCLUSION: These results suggest that UTP-induced relaxation is mediated by nucleotide receptors on endothelium and the contraction is mediated by pyrimidinoceptors on vascular smooth muscle.
Adenosine Triphosphate ; Animals ; Aorta ; Aorta, Thoracic* ; Endothelium ; Extracellular Space ; Guanosine Triphosphate ; Isometric Contraction ; Muscle, Smooth, Vascular ; Norepinephrine ; Rats* ; Receptors, Purinergic ; Receptors, Purinergic P2X ; Relaxation ; Suramin ; Transducers ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Monophosphate ; Uridine Triphosphate ; Uridine*

Mycophenolate mofetil(MMF) is a new immunosuppressant which non-competitively and reversibly blocks the de novo synthesis of guanine nucleotides required for DNA and RNA synthesis during lymphocyte proliferation. So MMF selectively inhibits lymphocyte proliferation and it has less side-effects than other immunosuppressants. We described 2 cases of generalized pustular psoriasis improved with MMF. We administered MMF combined with cyclosporin in case 1 and acitretin in case 2. Both patients markedly improved with MMF administration 3 days after and had no severe side-effects. Thus the authors thought that MMF appears to be an effective therapeutic alternative in the treatment of generalized pustular psoriasis.
Acitretin ; Cyclosporine ; DNA ; Guanine Nucleotides ; Humans ; Immunosuppressive Agents ; Lymphocytes ; Psoriasis* ; RNA

Tomato extract has been shown to exert antiplatelet activity in vitro and to change platelet function ex vivo, but with limitations. In this study, antiplatelet activity of water soluble tomato concentrate (Fruitflow I) and dry water soluble tomato concentrate (Fruitflow II) was investigated using rat platelets. Aggregation was induced by collagen and adenosine diphosphate and granule-secretion, [Ca2+]i, thromboxane B2, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels were examined. The activation of integrin αIIbβ3 and phosphorylation of signaling molecules, including mitogen-activated protein kinase (MAPK) and PI3K/Akt, were investigated by flow cytometry and immunoblotting, respectively. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were examined. Moreover, in vivo thrombus weight was tested by an arteriovenous shunt model. Fruitflow I and Fruitflow II significantly inhibited agonist induced platelet aggregation, adenosine triphosphate and serotonin release, [Ca2+]i, and thromboxane B2 concentration, while having no effect on cAMP and cGMP levels. Integrin αIIbβ3 activation was also significantly decreased. Moreover, both concentrates reduced phosphorylation of MAPK pathway factors such as ERK, JNK, P38, and PI3K/Akt. In vivo thrombus formation was also inhibited. Taken together, these concentrates have the potential for ethnomedicinal applications to prevent cardiovascular ailments and can be used as functional foods.
Adenosine Diphosphate ; Adenosine Monophosphate ; Adenosine Triphosphate ; Animals ; Blood Platelets* ; Cardiovascular Diseases ; Collagen ; Flow Cytometry ; Functional Food ; Guanosine Monophosphate ; Immunoblotting ; In Vitro Techniques ; Lycopersicon esculentum* ; Partial Thromboplastin Time ; Phosphorylation ; Platelet Aggregation ; Protein Kinases* ; Prothrombin Time ; Rats ; Serotonin ; Thrombosis ; Thromboxane B2 ; Water*

Guanine aminohydrolase(GAH;EC 3. 5. 4. 3.) was partially purified 122-fold from rat cerebrum to a specific activity of 7.22 in its per mg protein with a recovery of 7.47% by fractionation with ammonium sulfate, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and isoelectric focusing(pH4-6). The molecular weight of partially purified rat cerebral guanine aminohydrolase was estimated to be 110,000. But, in the cerebral cytosol, a rather higher molecular weight form of the enzyme was identified. The activity of the higher molecular weight form of guanine aminohydrolase was increased by dialyzing the cytosol, and it was converted into the lower molecular weight form(M.W.110,000) by addition of 2-mercaptoethanol. The reaction velocity of partially purified guanine aminohydrolase of rat cerebrum disclosed a hyperbolic curve, with its KM being 6.0uM at pH 8.0. The preparation showed high substrate specificity:among the purine nucleotides, nucleosides and bases with amino group, only guanosine and guanine were deaminated by the enzyme, and the reaction rate of the enzyme displayed by guanosine was less than 10% of that by guanine. When observed under the equimolar concentration of the substrate, hypoxanthine as well as inosine inhibited the activity of the rat cerebral guanine aminohydrolase by 9.4 and 7.8%, respectively, while 5-aminoimidazole-4-carboxamide inhibited the activity of it by 38%. The activity was inhibited by p-hydroxymercuric benzoate as well. Complete loss of its activity was observed after 30 minutes incubation at 60 degrees C, suggesting the preparation was heat labile.
Ammonium Sulfate ; Animals ; Benzoates ; Cerebrum* ; Chromatography ; Chromatography, Gel ; Cytosol ; DEAE-Cellulose ; Durapatite ; Filtration ; Guanine Deaminase* ; Guanine* ; Guanosine ; Hot Temperature ; Hydrogen-Ion Concentration ; Hypoxanthine ; Inosine ; Mercaptoethanol ; Molecular Weight ; Nucleosides ; Purine Nucleotides ; Rats* ; Xanthine Oxidase

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.
Cysteine/metabolism* ; Female ; GTP-Binding Proteins/metabolism* ; Guanine Nucleotides/pharmacology ; Human ; Methylation ; Placenta/metabolism* ; Placenta/enzymology ; Pregnancy ; Pregnancy Proteins/metabolism* ; Protein Methyltransferases/metabolism*

PURPOSE: 7-Bromomethylbenz[a]anthracene is a well- known mutagen and carcinogen. The aim of this study is to determine the mutagenic potency of its two major DNA adducts [N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[a]a2G) and N6-(benz[a]anthracen-7- ylmethyl)-2'-deoxyadenosine (b[a]a6A)] and the simpler benzylated analogs [N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A)] in Ad293 human cells and to compare to their mutagenicity in human cells and E. coli. MATERIALS AND METHODS: The shuttle vector pGP50 is capable of replicating in E. coli and human cells. Modified nucleotides were positioned in the plasmid pGP50 in a manner similar to pGP10 as described (8). Adenovirus transformed human embryonic kidney cells (line 293) were transfected with a shuttle vector containing an adduct. Two days later, the plasmids were recovered and treated with DpnI to remove unreplicated DNA. DH10B E. coli were transformed with the plasmids. Bacteria were cultured with the media containing X-gal, IPTG and ampicillin. Bacteria transformed by the plasmid with the adduct-induced mutation in the initiation codon of lacZ' form white colonies whereas bacteria transformed by the plasmid without mutation form blue colonies. RESULTS: In the human cell site-specific mutagenesis system, bn2G exhibited weak mutagenicity and bn6A was not mutagenic, although b[a]a2G or b[a]a6A produced 8% and 7% mutant colonies, respectively. At the site of the adduct, b[a]a2G induced the G--> T transversion mutation while b[a]a6A produced the A--> G transition mutation. CONCLUSION: These data indicate that bulkier b[a]a2G and b[a]a6A exhibit significantly greater mutagenicity in human cells than in E. coli.
Adenine* ; Adenoviridae ; Ampicillin ; Bacteria ; Codon, Initiator ; DNA ; DNA Adducts ; Genetic Vectors ; Guanine* ; Humans* ; Isopropyl Thiogalactoside ; Kidney ; Mutagenesis ; Mutagenesis, Site-Directed* ; Nucleotides ; Plasmids

Objective: To analyze guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) expression based on bioinformatics, so as to evaluate its role and its relationship with survival rate during the occurrence and development of hepatocellular carcinoma. Methods: GEPIA, UALCAN and HPA databases were used to analyze the expression level of GNB2L1 and its relationship with HCC survival rate. Mutations in the GNB2L1 gene and their impact on survival were analyzed using the cBioPortal database. LinkedOmics database was used to analyze GNB2L1-related genes in HCC. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed simultaneously. STEING database was used to construct the GNB2L1 protein interaction network. TIMER database was used to analyze the relationship between GNB2L1 gene expression and immune infiltration in hepatocellular carcinoma. Differential expression of GNB2L1 in plasma platelets of HCC patients and healthy controls was analyzed using mRNA-based sequencing technology. Data between groups were compared using an independent-samples t-test. Results: GNB2L1 expression level was significantly increased in HCC tissues (P<0.05), and its expression was significantly correlated with body weight, classification and stage (P<0.05). The overall survival rate was higher in GNB2L1 low expression group (P<0.001). GNB2L1 and its related genes were related to biological process regulation, metabolic process, protein binding, oxidative phosphorylation, JAK-STAT signaling pathway, Ras signaling pathway and so on. GNB2L1 had interaction with RPS12, RPS11 and RPL19, and participated in multiple biological processes such as liver regeneration and positive regulation of endogenous apoptotic signaling pathway. GNB2L1 expression was significantly positively correlated with the infiltration degree of various immune cells in HCC (P<0.05). Cox regression analysis showed that GNB2L1 was an independent risk factor for lower survival rate in patients with HCC [Hazard ratio (95% confidence interval)=1.456 (1.034~2.051), P=0.031]. GNB2L1expression levels were significantly higher in platelets of HCC patients than that of healthy controls (10.40±1.36 vs. 9.58±0.51, t=2.194, P=0.037). Conclusion: GNB2L1 has high expression and close relationship to survival rate in HCC. Therefore, GNB2L1 may be a potential biomarker of HCC.
Humans ; Carcinoma, Hepatocellular/pathology* ; Computational Biology ; Liver Neoplasms/pathology* ; Protein Subunits/metabolism* ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; RNA, Messenger ; Guanine Nucleotides ; Gene Expression ; Biomarkers, Tumor/genetics*

PURPOSE: This study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients. MATERIALS AND METHODS: One hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring. RESULTS: The result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4+/-0.31 mg/kg/day before monitoring and 1.1+/-0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring. CONCLUSION: TPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.
Adolescent ; Adult ; Azathioprine/*adverse effects/therapeutic use ; Child ; Child, Preschool ; Female ; Genotype ; Guanine Nucleotides/metabolism ; Humans ; Inflammatory Bowel Diseases/*drug therapy/metabolism ; Male ; Methyltransferases/genetics/metabolism ; Republic of Korea ; Risk Factors ; Thionucleotides/metabolism ; Treatment Outcome

PURPOSE: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), is the active metabolite of the immunosuppressive drug, mycophenolate mofetil (MMF). MMF is used to prevent an immune- mediate rejection response following organ transplantation via the inhibition of the IMPDH and GTP biosynthesis pathway. This study was designed to elucidate the mechanism by which MPA exerts its cytotoxic effect on human T lymphocytic and monocytic cell lines. METHODS: MOLT-4 and U937 cell lines were treated with MPA. Cell viability, expression of Bcl2 family proteins and Fas/Fas-L, effects of antioxidants and intracellular Ca2+ regulating agents and apoptosis were measured using a variety of microscopic and biochemical techniques. RESULTS: MPA induced the death of U937 and MOLT-4 cells in dose and time dependent manners, which was revealed an apoptosis with a characteristic ladder pattern of DNA fragmentation. In addition, BAPTA/AM, an intracellular Ca2+ chelator protected MOLT-4 cells from MPA treated apoptosis, although it did not have an additive with thapsigargin, and increases cytosolic Ca2+ stores. However, antioxidants including reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) did not inhibit the apoptosis of cells by MPA. Furthermore, guanosine suppressed MPA induced apoptosis of MOLT-4 lymphocytes, although adenosine did not. MPA also increased the catalytic activity of caspase family cysteine proteases including caspase-8, 9 and 3 proteases in MOLT-4 cells. Sequential activation indicated that the cleavage of caspase-8 and 9 precedes those of caspase-3. CONCLUSION: The results suggest that MPA induces the apoptotic death of MOLT-4 lymphocytes via the activations of caspase family proteases and the depletion of GTP.
Acetylcysteine ; Adenosine ; Antioxidants ; Apoptosis ; Caspase 3 ; Caspase 8 ; Cell Line ; Cell Survival ; Cysteine Proteases ; Cytosol ; DNA Fragmentation ; Glutathione ; Guanosine ; Guanosine Triphosphate ; Humans ; Inosine Monophosphate ; Lymphocytes ; Mycophenolic Acid* ; Organ Transplantation ; Oxidoreductases ; Peptide Hydrolases ; Signal Transduction* ; T-Lymphocytes* ; Thapsigargin ; Transplants ; U937 Cells