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Molecular analysis of mouse Ampd3 gene encoding heart-type isoform of AMP deaminase
9TH INTERNATIONAL/6TH EUROPEAN J O I N T SYMPOSIUM ON PURINE AND PYRIMIDINE METABOLISM IN MAN
Results MPA induces time- and dose-dependent inhibition of
Conclusion Activation of RAR~ gene by demethylation is respon-
proliferation, decrease in GTP pools, arrest at the G1/S interphase a n d cell d e a t h by apoptosis in all cell lines. In LA-N-5 the fraction of apoptotic cells was about 40% at 1 ~M MPA after 48 hours of t r e a t m e n t . Apoptosis was associated with up regulation of p53 and down regulation of Bcl-2 expression. The effects of MPA were, at least partially, reversed by the addition of guanine or guanosine to the culture medium. Conclusion MPA induces apoptosis in h u m a n neuroblastoma cell lines, suggesting a potential clinical use of the drug.
sible in p a r t for the a n t i t u m o r action of 5-AZA and increased tumor sensitivity to RA.
112 A N HPLC-LINKED NON-RADIOCHEMICAL ASSAY OF OPRT A N D ODC ACTIVITY IN ERYTHROCYTES: ONE CASE OF OROTIC ACIDURIA V. Mieheli,* G. Jaeomelli*, E. Z a m m a r c h i ° and G. Pompucci* *Dip. Biologia Molecolare--Universit~ di Siena; °Ospedale Pediatrico Mayer, Firenze--ITALIA
Objectives One or both Orotate phosphoribosyl-transferase (OPRT) and OMP decarboxylase (ODC) activities, responsible for U M P synthesis, are known to be defective in h u m a n m u t a n t s with orotic aciduria. We present a n HPLC-linked, non-radiochemical assay method for both activities in erythrocytes, based on the direct m e a s u r e m e n t of products, OMP a n d UMP. Design and Method 15 control children, aged 4 - 9 , and a 9 year old boy, presenting with orotic aciduria and mild m e n t a l retardation, were examined. Lysates from 100 ~L1of packed erythrocytes were used to assay both OPRT a n d ODC activities and haemoglobin concentration. Substrates and products were separated and quantified by RF-HPLC (System Gold Beckman, LC-18 Supelcosil column; elution time 15 min) in perchloric extracts of the incubation mixtures. Results and Discussion The OPRT activity in controls was 0.45 ± 0.19 nmoles/h/mg Hb, ODC activity was 0.40 ± 0.17. Both activities were markedly lower in the oroticaciduric child (0.072 and 0.04, respectively), The non-radiochemical HPLC method presented is easy to perform, sensitive and reliable enough to be used for diagnostic purposes.
113 5-AZADEOXYCYTIDINE: A MODEL AGENT TO STUDY IMPACT OF DIFFERENTIATION IN CANCER CHEMOTHERAPY Momparler, R.L. a n d C6t~, S. Dept. pharmacologie, Universit~ de Montreal & Centre de recherche, H6pital SteJustine, Montreal, Quebec H3T 1C5, C a n a d a 5-Azadeoxycytidine (5-AZA) can induce differentiation of cancer cells by blocking methylation of cytosine in DNA leading to activation of specific genes. Tumor suppressor genes t h a t have been silenced by methylation are potential targets for 5-AZA therapy. Objectives To determine if 5-AZA can activate the expression of the retinoic acid receptor ~ (RARe), a putative t u m o r suppressor gene, in t u m o r cells. Methods H u m a n DLD-1 colon carcinoma cells were treated with 5-AZA and/or all-trans retinoic acid (RA) a n d cell survival determined by colony assay. The activation of RAR~ expression by 5-AZA was determined by RT-PCR a n d N o r t h e r n blotting. Demethylation of the RAR~ gene was determined by S o u t h e r n blotting after digestion with methylation-sensitive restriction enzymes. Results 5-AZA activated the expression of RAR~ gene in DLD-1 colon t u m o r cells by demethylation of CpG islands present in the promoter region of this gene.
270
114 MOLECULAR ANALYSIS OF MOUSE Ampd3 GENE ENCODING HEART-TYPE ISOFORM OF AMP DEAMINASE Takayuki Morisaki, Kannika Sermsuvitayawong, Xudong Wang, Akira Nagabukuro 1, Yoichi M a t s u d a 1, Nobuaki Ogasawara 2, Ikuo Mineo 3, Hiroko Morisaki, Tsunehiro Mukai. Dept Bioscienee, Natl Cardiovasc Ctr Res Inst, 1Nagoya Univ, 2Aichi Pref Colony, and 3Otemae Hosp
Objectives We have studied murine gene for the heart-type (H) isoform of AMP deaminase (AMPD) since this isoform has not been well understood in the molecular level. Design and Methods RT-PCR and screening of cDNA and genomic DNA libraries were employed to isolate the mouse cDNA and gene for H-AMPD. Molecular characterization including prokaryotic expression of the isolated cDNA was performed. Results Three mouse genes for AMPD were disclosed. Mouse Ampd3 cDNA was isolated and found to encode a 766 amino acid peptide which was confirmed as H-AMPD. Ampd3 gene was also isolated, and it was mapped in mouse chromosome 7. The 5' non-coding and the promoter regions of mouse Ampd3 are not similar to those of h u m a n AMPD3, although these two genes are homologous. Different tissue distribution of the transcripts were found in these genes. Prokaryotic expressed peptides showed different characters in the mouse and h u m a n AMPD3 peptides. Conclusion Murine H-type isoform of AMPD is encoded inAmpd3 gene, which is syntenic to h u m a n AMPD3. However, mouse Ampd3 is degenerated from h u m a n AMPD3.
115 ZONAL DISTRIBUTION OF ALLOPURINOL-OXIDIZING ENZYMES IN RAT LIVER Moriwaki, Y., Yamamoto, T., Yamakita, J., Takahashi, S., Tsutsumi, Z., and Higashino, K., Third D e p a r t m e n t of I n t e r n a l Medicine, Hyogo College of Medicine Mukogawacho 1-1, Nishinomiya, Hyogo 663, J a p a n
Objectives To demonstrate the lobular distribution of allopurinoloxidizing enzymes, x a n t h i n e and aldehyde oxidase activities in r a t liver. Design and Methods The enzyme histochemical localization of the enzymes was performed by tetrazolium method, using unfixed cryostat section of the liver from male Wistar rat. Results The method demonstrated both oxidases in the cytoplasm of liver cells. The distribution of the both enzyme activities in the liver was uneven, more p r e d o m i n a n t in the pericentral t h a n in the periportal area. Conclusion The result was consistent with immunohistochemical localization study of the enzymes. The possible pathophysiological role of these enzymes were discussed in light of the lobular distribution of the enzymes in the liver.
116 COMPARISON OF PYRIMIDINE NUCLEOSIDE SALVAGE ENZYMES IN DROSOPHILA M E L A N O GASTER AND MAN Munch-Petersen, B.*, Piskur, J., and Sondergaard, L. *Dept. Life Sci. Chem. Roskilde University, P.O. BOX 260, 4000 Roskilde, D e n m a r k
Objectives To compare pyrimidine salvage in eucaryotic cells. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
Results MPA induces time- and dose-dependent inhibition of
Conclusion Activation of RAR~ gene by demethylation is respon-
proliferation, decrease in GTP pools, arrest at the G1/S interphase a n d cell d e a t h by apoptosis in all cell lines. In LA-N-5 the fraction of apoptotic cells was about 40% at 1 ~M MPA after 48 hours of t r e a t m e n t . Apoptosis was associated with up regulation of p53 and down regulation of Bcl-2 expression. The effects of MPA were, at least partially, reversed by the addition of guanine or guanosine to the culture medium. Conclusion MPA induces apoptosis in h u m a n neuroblastoma cell lines, suggesting a potential clinical use of the drug.
sible in p a r t for the a n t i t u m o r action of 5-AZA and increased tumor sensitivity to RA.
112 A N HPLC-LINKED NON-RADIOCHEMICAL ASSAY OF OPRT A N D ODC ACTIVITY IN ERYTHROCYTES: ONE CASE OF OROTIC ACIDURIA V. Mieheli,* G. Jaeomelli*, E. Z a m m a r c h i ° and G. Pompucci* *Dip. Biologia Molecolare--Universit~ di Siena; °Ospedale Pediatrico Mayer, Firenze--ITALIA
Objectives One or both Orotate phosphoribosyl-transferase (OPRT) and OMP decarboxylase (ODC) activities, responsible for U M P synthesis, are known to be defective in h u m a n m u t a n t s with orotic aciduria. We present a n HPLC-linked, non-radiochemical assay method for both activities in erythrocytes, based on the direct m e a s u r e m e n t of products, OMP a n d UMP. Design and Method 15 control children, aged 4 - 9 , and a 9 year old boy, presenting with orotic aciduria and mild m e n t a l retardation, were examined. Lysates from 100 ~L1of packed erythrocytes were used to assay both OPRT a n d ODC activities and haemoglobin concentration. Substrates and products were separated and quantified by RF-HPLC (System Gold Beckman, LC-18 Supelcosil column; elution time 15 min) in perchloric extracts of the incubation mixtures. Results and Discussion The OPRT activity in controls was 0.45 ± 0.19 nmoles/h/mg Hb, ODC activity was 0.40 ± 0.17. Both activities were markedly lower in the oroticaciduric child (0.072 and 0.04, respectively), The non-radiochemical HPLC method presented is easy to perform, sensitive and reliable enough to be used for diagnostic purposes.
113 5-AZADEOXYCYTIDINE: A MODEL AGENT TO STUDY IMPACT OF DIFFERENTIATION IN CANCER CHEMOTHERAPY Momparler, R.L. a n d C6t~, S. Dept. pharmacologie, Universit~ de Montreal & Centre de recherche, H6pital SteJustine, Montreal, Quebec H3T 1C5, C a n a d a 5-Azadeoxycytidine (5-AZA) can induce differentiation of cancer cells by blocking methylation of cytosine in DNA leading to activation of specific genes. Tumor suppressor genes t h a t have been silenced by methylation are potential targets for 5-AZA therapy. Objectives To determine if 5-AZA can activate the expression of the retinoic acid receptor ~ (RARe), a putative t u m o r suppressor gene, in t u m o r cells. Methods H u m a n DLD-1 colon carcinoma cells were treated with 5-AZA and/or all-trans retinoic acid (RA) a n d cell survival determined by colony assay. The activation of RAR~ expression by 5-AZA was determined by RT-PCR a n d N o r t h e r n blotting. Demethylation of the RAR~ gene was determined by S o u t h e r n blotting after digestion with methylation-sensitive restriction enzymes. Results 5-AZA activated the expression of RAR~ gene in DLD-1 colon t u m o r cells by demethylation of CpG islands present in the promoter region of this gene.
270
114 MOLECULAR ANALYSIS OF MOUSE Ampd3 GENE ENCODING HEART-TYPE ISOFORM OF AMP DEAMINASE Takayuki Morisaki, Kannika Sermsuvitayawong, Xudong Wang, Akira Nagabukuro 1, Yoichi M a t s u d a 1, Nobuaki Ogasawara 2, Ikuo Mineo 3, Hiroko Morisaki, Tsunehiro Mukai. Dept Bioscienee, Natl Cardiovasc Ctr Res Inst, 1Nagoya Univ, 2Aichi Pref Colony, and 3Otemae Hosp
Objectives We have studied murine gene for the heart-type (H) isoform of AMP deaminase (AMPD) since this isoform has not been well understood in the molecular level. Design and Methods RT-PCR and screening of cDNA and genomic DNA libraries were employed to isolate the mouse cDNA and gene for H-AMPD. Molecular characterization including prokaryotic expression of the isolated cDNA was performed. Results Three mouse genes for AMPD were disclosed. Mouse Ampd3 cDNA was isolated and found to encode a 766 amino acid peptide which was confirmed as H-AMPD. Ampd3 gene was also isolated, and it was mapped in mouse chromosome 7. The 5' non-coding and the promoter regions of mouse Ampd3 are not similar to those of h u m a n AMPD3, although these two genes are homologous. Different tissue distribution of the transcripts were found in these genes. Prokaryotic expressed peptides showed different characters in the mouse and h u m a n AMPD3 peptides. Conclusion Murine H-type isoform of AMPD is encoded inAmpd3 gene, which is syntenic to h u m a n AMPD3. However, mouse Ampd3 is degenerated from h u m a n AMPD3.
115 ZONAL DISTRIBUTION OF ALLOPURINOL-OXIDIZING ENZYMES IN RAT LIVER Moriwaki, Y., Yamamoto, T., Yamakita, J., Takahashi, S., Tsutsumi, Z., and Higashino, K., Third D e p a r t m e n t of I n t e r n a l Medicine, Hyogo College of Medicine Mukogawacho 1-1, Nishinomiya, Hyogo 663, J a p a n
Objectives To demonstrate the lobular distribution of allopurinoloxidizing enzymes, x a n t h i n e and aldehyde oxidase activities in r a t liver. Design and Methods The enzyme histochemical localization of the enzymes was performed by tetrazolium method, using unfixed cryostat section of the liver from male Wistar rat. Results The method demonstrated both oxidases in the cytoplasm of liver cells. The distribution of the both enzyme activities in the liver was uneven, more p r e d o m i n a n t in the pericentral t h a n in the periportal area. Conclusion The result was consistent with immunohistochemical localization study of the enzymes. The possible pathophysiological role of these enzymes were discussed in light of the lobular distribution of the enzymes in the liver.
116 COMPARISON OF PYRIMIDINE NUCLEOSIDE SALVAGE ENZYMES IN DROSOPHILA M E L A N O GASTER AND MAN Munch-Petersen, B.*, Piskur, J., and Sondergaard, L. *Dept. Life Sci. Chem. Roskilde University, P.O. BOX 260, 4000 Roskilde, D e n m a r k
Objectives To compare pyrimidine salvage in eucaryotic cells. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997