Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1139126
Juan Manuel Orozco Rodriguez, Mohamad Nesrini, L. S. Christiansen, W. Knecht
ABSTRACT Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure–function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.
{"title":"Expression of tomato thymidine kinase 1 by means of the baculovirus expression vector system","authors":"Juan Manuel Orozco Rodriguez, Mohamad Nesrini, L. S. Christiansen, W. Knecht","doi":"10.1080/15257770.2016.1139126","DOIUrl":"https://doi.org/10.1080/15257770.2016.1139126","url":null,"abstract":"ABSTRACT Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure–function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"15 1","pages":"691 - 698"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76852131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1216566
R. Honeywell, Dzjemma Sarkisjan, I. Kathmann, M. H. Kristensen, G. Peters
ABSTRACT Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6–6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.
{"title":"Sensitive liquid chromatography mass spectrometry (LC-MS) assay reveals novel insights on DNA methylation and incorporation of gemcitabine, its metabolite difluorodeoxyuridine, deoxyuridine, and RX-3117 into DNA","authors":"R. Honeywell, Dzjemma Sarkisjan, I. Kathmann, M. H. Kristensen, G. Peters","doi":"10.1080/15257770.2016.1216566","DOIUrl":"https://doi.org/10.1080/15257770.2016.1216566","url":null,"abstract":"ABSTRACT Antimetabolites are incorporated into DNA and RNA, affecting their function. Liquid-chromatography-mass-spectrometry (LC-MS-MS) permits the sensitive, selective analysis of normal nucleosides. The method was adapted to measure the incorporation of deoxyuridine, gemcitabine (difluorodeoxycytidine), its metabolite difluorodeoxyuridine (dFdU), and the novel compound fluorocyclopentenylcytosine (RX3117). DNA was degraded to its deoxynucleotides for quantification by LC-MS-MS, gradient chromatography on a Phenomenex prodigy-3-ODS with positive ionization. The range of deoxyuridine DNA-mis-incorporation varied nine-fold in 27 cell lines (leukemia, colon, ovarian, lung cancer). At low-folate conditions a 2.1-fold increase in deoxyuridine was observed. Global methylation (given as % 5-methyl-deoxycytidine) was comparable between the cell lines (4.6–6.5%). Exposure of A2780 cells to 1 μM gemcitabine (4 hours) resulted in 3.6 pmol gemcitabine/μg DNA, but in AG6000 cells (deoxycytidine-kinase-deficient) no incorporation was found. However, when A2780, AG6000, or CCRF-CEM cells were exposed to 100 μM dFdU we found it as gemcitabine, 20.5, 19.6, and 0.51 pmol gemcitabine/μg DNA, respectively. Preincubation of CCRF-CEM cells with cyclopentenyl-cytosine (a CTP-synthetase inhibitor) increased dFdU incorporation four-fold. Apparently dFdU is activated independently of deoxycytidine-kinase and possibly converted in-situ to dFdCMP. RX3117 was incorporated into both DNA and RNA (0.0037 and 0.00515 pmol/μg, respectively). In summary, a sensitive method to quantify the incorporation of gemcitabine, deoxyuridine, and RX-3117 was developed, which revealed that dFdU was incorporated into DNA as the parent compound gemcitabine.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"24 1","pages":"652 - 662"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74265438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1163379
Michito Nagura, Yoshifuru Tamura, T. Kumagai, M. Hosoyamada, S. Uchida
ABSTRACT Uric acid (UA) is a potential risk factor of the progression of chronic kidney disease (CKD). Recently, we reported that intestinal UA excretion might be enhanced via upregulation of the ATP-binding cassette transporter G2 (Abcg2) in a 5/6 nephrectomy (Nx) rat model. In the present study, we examined the mRNA and protein expressions of UA transporters, URAT1, GLUT9/URATv1, ABCG2 and NPT4 in the kidney and ileum in the same rat model. Additionally, we investigated the Abcg2 mRNA expression of ileum in hyperuricemic rat model by orally administering oxonic acid. Male Wistar rats were randomly assigned to three groups consisting of Nx group, oxonic acid-treated (Ox) group and sham-operated control group, and sacrificed at 8 weeks. Creatinine and UA were measured and the mRNA expressions of UA transporters in the kidney and intestine were evaluated by a real time PCR. UA transporters in the kidney sections were also examined by immunohistochemistry. Serum creatinine elevated in the Nx group whereas serum UA increased in the Ox group. Both the mRNA expression and the immunohistochemistry of the UA transporters were decreased in the Nx group, suggesting a marginal role in UA elevation in decreased kidney function. In contrast, the mRNA expression of Abcg2 in the ileum significantly increased in the Ox group. These results suggest that the upregulation of Abcg2 mRNA in the ileum triggered by an elevation of serum UA may play a compensatory role in increasing intestinal UA excretion.
{"title":"Uric acid metabolism of kidney and intestine in a rat model of chronic kidney disease","authors":"Michito Nagura, Yoshifuru Tamura, T. Kumagai, M. Hosoyamada, S. Uchida","doi":"10.1080/15257770.2016.1163379","DOIUrl":"https://doi.org/10.1080/15257770.2016.1163379","url":null,"abstract":"ABSTRACT Uric acid (UA) is a potential risk factor of the progression of chronic kidney disease (CKD). Recently, we reported that intestinal UA excretion might be enhanced via upregulation of the ATP-binding cassette transporter G2 (Abcg2) in a 5/6 nephrectomy (Nx) rat model. In the present study, we examined the mRNA and protein expressions of UA transporters, URAT1, GLUT9/URATv1, ABCG2 and NPT4 in the kidney and ileum in the same rat model. Additionally, we investigated the Abcg2 mRNA expression of ileum in hyperuricemic rat model by orally administering oxonic acid. Male Wistar rats were randomly assigned to three groups consisting of Nx group, oxonic acid-treated (Ox) group and sham-operated control group, and sacrificed at 8 weeks. Creatinine and UA were measured and the mRNA expressions of UA transporters in the kidney and intestine were evaluated by a real time PCR. UA transporters in the kidney sections were also examined by immunohistochemistry. Serum creatinine elevated in the Nx group whereas serum UA increased in the Ox group. Both the mRNA expression and the immunohistochemistry of the UA transporters were decreased in the Nx group, suggesting a marginal role in UA elevation in decreased kidney function. In contrast, the mRNA expression of Abcg2 in the ileum significantly increased in the Ox group. These results suggest that the upregulation of Abcg2 mRNA in the ileum triggered by an elevation of serum UA may play a compensatory role in increasing intestinal UA excretion.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"1 1","pages":"550 - 558"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89434831","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1200074
M. Zafar, Z. Naydenova, I. Coe
ABSTRACT Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.
{"title":"Extended exposure to substrate regulates the human equilibrative nucleoside transporter 1 (hENT1)","authors":"M. Zafar, Z. Naydenova, I. Coe","doi":"10.1080/15257770.2016.1200074","DOIUrl":"https://doi.org/10.1080/15257770.2016.1200074","url":null,"abstract":"ABSTRACT Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"29 1","pages":"631 - 642"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80462923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1149192
M. Sakiyama, H. Matsuo, S. Nagamori, W. Ling, Y. Kawamura, A. Nakayama, T. Higashino, T. Chiba, K. Ichida, Y. Kanai, N. Shinomiya
ABSTRACT Human sodium-dependent phosphate cotransporter type 1 (NPT1/SLC17A1) is one of the urate transporters in the kidney. Our recent study revealed that a common missense variant, I269T (rs1165196), of NPT1 decreases the risk of renal underexcretion gout. Moreover, we demonstrated that human NPT1 is localized to the apical membrane of the renal proximal tubule, and that I269T is the gain-of-function variant which increases the NPT1-mediated urate export. However, the mechanism by which I269T variant increases the urate export remains to be clarified. Thus, we performed immunostaining and functional analysis of human NPT1 using the Xenopus oocyte expression system. For comparison of human NPT1 expression levels of oocyte membrane between 269I (wild type) and 269T (variant), immunostaining was performed with anti-human NPT1 antibodies. As a result, we showed that NPT1 I269T variant did not change the human NPT1 membrane expression levels, although NPT1 I269T variant increased the urate transport compared with NPT1 wild type. Combined with the previous report that I269T variant did not induce Km changes but increased the Vmax of urate transport in a proteoliposome system, our findings suggest that I269T variant increases NPT1-mediated urate export without increase of NPT1 expression levels on the membrane. Thus, I269T, a common missense variant of NPT1, might have faster conformation changes than NPT1 wild type in terms of the alternating-access model of transporters, and increases renal urate export in humans.
{"title":"Expression of a human NPT1/SLC17A1 missense variant which increases urate export","authors":"M. Sakiyama, H. Matsuo, S. Nagamori, W. Ling, Y. Kawamura, A. Nakayama, T. Higashino, T. Chiba, K. Ichida, Y. Kanai, N. Shinomiya","doi":"10.1080/15257770.2016.1149192","DOIUrl":"https://doi.org/10.1080/15257770.2016.1149192","url":null,"abstract":"ABSTRACT Human sodium-dependent phosphate cotransporter type 1 (NPT1/SLC17A1) is one of the urate transporters in the kidney. Our recent study revealed that a common missense variant, I269T (rs1165196), of NPT1 decreases the risk of renal underexcretion gout. Moreover, we demonstrated that human NPT1 is localized to the apical membrane of the renal proximal tubule, and that I269T is the gain-of-function variant which increases the NPT1-mediated urate export. However, the mechanism by which I269T variant increases the urate export remains to be clarified. Thus, we performed immunostaining and functional analysis of human NPT1 using the Xenopus oocyte expression system. For comparison of human NPT1 expression levels of oocyte membrane between 269I (wild type) and 269T (variant), immunostaining was performed with anti-human NPT1 antibodies. As a result, we showed that NPT1 I269T variant did not change the human NPT1 membrane expression levels, although NPT1 I269T variant increased the urate transport compared with NPT1 wild type. Combined with the previous report that I269T variant did not induce Km changes but increased the Vmax of urate transport in a proteoliposome system, our findings suggest that I269T variant increases NPT1-mediated urate export without increase of NPT1 expression levels on the membrane. Thus, I269T, a common missense variant of NPT1, might have faster conformation changes than NPT1 wild type in terms of the alternating-access model of transporters, and increases renal urate export in humans.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"304 1","pages":"536 - 542"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77350627","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1154970
M. Zabielska, B. Kutryb-Zając, Paulina Żukowska, E. Slominska, R. Smolenski
ABSTRACT 4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. Methods. Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. Results. Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm2 in C to 16.0±2.6 nmol/min/cm2 in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. Conclusions. This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.
{"title":"Effects of 4-Pyridone-3-carboxamide-1β-D-ribonucleoside on adenine nucleotide catabolism in the aortic wall; Implications for atherosclerosis in ApoE-/-LDLR-/- mice","authors":"M. Zabielska, B. Kutryb-Zając, Paulina Żukowska, E. Slominska, R. Smolenski","doi":"10.1080/15257770.2016.1154970","DOIUrl":"https://doi.org/10.1080/15257770.2016.1154970","url":null,"abstract":"ABSTRACT 4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. Methods. Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. Results. Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm2 in C to 16.0±2.6 nmol/min/cm2 in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. Conclusions. This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"77 1","pages":"720 - 725"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84963302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1154969
Marta Toczek, B. Kutryb-Zając, Paulina Żukowska, E. Slominska, M. Isalan, M. Mielcarek, R. Smolenski
ABSTRACT Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.
{"title":"Changes in cardiac nucleotide metabolism in Huntington's disease","authors":"Marta Toczek, B. Kutryb-Zając, Paulina Żukowska, E. Slominska, M. Isalan, M. Mielcarek, R. Smolenski","doi":"10.1080/15257770.2016.1154969","DOIUrl":"https://doi.org/10.1080/15257770.2016.1154969","url":null,"abstract":"ABSTRACT Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"43 1","pages":"707 - 712"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86472374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1163377
Ewa Kaniewska-Bednarczuk, M. Mielcarek, A. Chester, E. Slominska, M. Yacoub, R. Smolenski
ABSTRACT Extracellular nucleotides regulate thrombosis, inflammation, and immune response. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) convert extracellular nucleotides in a sequential order: ATP to ADP, AMP, and then to adenosine. In this study, we aimed to test an effect of oxidized low-density lipoprotein (ox-LDL) on CD39 and CD73 in endothelial cells. Human aortic valve endothelial cells were exposed to ox-LDL for 24–48 h. Next, the activity, protein expression, and mRNA transcripts level of CD39 and CD73 were characterized by an incubation with ATP or AMP followed by high-performance liquid chromatography analysis of media as well as western blots and qPCR. CD73 activity in human valve endothelial cells was increased in presence of ox-LDL (4.04 ± 0.32 nmol/mg prot./min, mean +/− SEM) as compared with control (2.75 ± 0.21 nmol/mg prot/min). There was almost no effect of ox-LDL on CD39 activity. A similar effect was observed for mRNA and protein expression. In conclusion, we found that ox-LDL modulated CD39 and CD73 activity in the endothelium, which may contribute to relevant pathologies and featured treatments.
{"title":"Oxidized low-density lipoproteins enhance expression and activity of CD39 and CD73 in the human aortic valve endothelium","authors":"Ewa Kaniewska-Bednarczuk, M. Mielcarek, A. Chester, E. Slominska, M. Yacoub, R. Smolenski","doi":"10.1080/15257770.2016.1163377","DOIUrl":"https://doi.org/10.1080/15257770.2016.1163377","url":null,"abstract":"ABSTRACT Extracellular nucleotides regulate thrombosis, inflammation, and immune response. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) convert extracellular nucleotides in a sequential order: ATP to ADP, AMP, and then to adenosine. In this study, we aimed to test an effect of oxidized low-density lipoprotein (ox-LDL) on CD39 and CD73 in endothelial cells. Human aortic valve endothelial cells were exposed to ox-LDL for 24–48 h. Next, the activity, protein expression, and mRNA transcripts level of CD39 and CD73 were characterized by an incubation with ATP or AMP followed by high-performance liquid chromatography analysis of media as well as western blots and qPCR. CD73 activity in human valve endothelial cells was increased in presence of ox-LDL (4.04 ± 0.32 nmol/mg prot./min, mean +/− SEM) as compared with control (2.75 ± 0.21 nmol/mg prot/min). There was almost no effect of ox-LDL on CD39 activity. A similar effect was observed for mRNA and protein expression. In conclusion, we found that ox-LDL modulated CD39 and CD73 activity in the endothelium, which may contribute to relevant pathologies and featured treatments.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"20 1","pages":"713 - 719"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87486890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2016.1143557
Zeeshan Mutahir, L. S. Christiansen, A. Clausen, M. Berchtold, Z. Gojković, B. Munch‐Petersen, W. Knecht, J. Piškur
ABSTRACT Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of dCK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters of the dCK/dGK enzymes encoded by these genes. The two dCK enzymes in G. gallus have broader substrate specificity than their human or X. laevis counterparts. Additionally, the duplicated dCK enzyme in G. gallus might have become mitochondria. Based on our study we postulate that changing and adapting substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs.
{"title":"Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family","authors":"Zeeshan Mutahir, L. S. Christiansen, A. Clausen, M. Berchtold, Z. Gojković, B. Munch‐Petersen, W. Knecht, J. Piškur","doi":"10.1080/15257770.2016.1143557","DOIUrl":"https://doi.org/10.1080/15257770.2016.1143557","url":null,"abstract":"ABSTRACT Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of dCK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters of the dCK/dGK enzymes encoded by these genes. The two dCK enzymes in G. gallus have broader substrate specificity than their human or X. laevis counterparts. Additionally, the duplicated dCK enzyme in G. gallus might have become mitochondria. Based on our study we postulate that changing and adapting substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"174 1","pages":"677 - 690"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83538180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-12-01DOI: 10.1080/15257770.2015.1131295
L. S. Christiansen, Gabriella C. van Zanten, D. Berenstein, Marianne Lauridsen, S. Kjærulff, L. Søndergaard, B. Munch‐Petersen
ABSTRACT We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3′-5′-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.
{"title":"Isolation of a novel protein, P12—from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3′-5′- exonuclease activity","authors":"L. S. Christiansen, Gabriella C. van Zanten, D. Berenstein, Marianne Lauridsen, S. Kjærulff, L. Søndergaard, B. Munch‐Petersen","doi":"10.1080/15257770.2015.1131295","DOIUrl":"https://doi.org/10.1080/15257770.2015.1131295","url":null,"abstract":"ABSTRACT We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3′-5′-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"1 1","pages":"699 - 706"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89899511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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