Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methylguanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromato-graphically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species was progressively converted to a co-dominant species that has a 4.5-min column retention. Mass spectroscopy confirmed the existence of two species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom7Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. These results demonstrate that the enzyme excises formylated rom7Gua from DNA Analysis of rom7Guo by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom7Gua.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23913/1/0000156.pd

Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives

Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom7Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom7Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-14C]guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH4H2PO4 (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH4H2PO4 (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, about 90% of the material was formylated. When stored at 24[deg]C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom7Gua are in equilibrium. The rom7Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23912/1/0000155.pd

Nicotinamide Inhibits Alkylating Agent-Induced Apoptotic Neurodegeneration in the Developing Rat Brain

BACKGROUND: Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain. METHODOLOGY/PRINCIPAL FINDINGS: Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment. CONCLUSION: Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants

Lack of EGF receptor contributes to drug sensitivity of human germline cells

Germline mutations have been associated with generation of various types of tumour. In this study, we investigated genetic alteration of germline tumours that affect the drug sensitivity of cells. Although all germline tumour cells we tested were hypersensitive to DNA-damaging drugs, no significant alteration was observed in their DNA repair activity or the expression of DNA repair proteins. In contrast, germline tumours expressed very low level of epidermal growth factor receptor (EGFR) compared to drug-resistant ovarian cancer cells. An immunohistochemical analysis indicated that most of the primary germline tumours we tested expressed very low level of EGFR. In accordance with this, overexpression of EGFR in germline tumour cells showed an increase in drug resistance, suggesting that a lack of EGFR, at least in part, contributes to the drug sensitivity of germline tumours

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine

A major and previously undetected carcinogen-DNA adduct was found in the livers of rats given N,N-dimethylnitrosamine or 1,2-dimethylhydrazine. This adduct, which accounted for 55% of the total methyl residues in DNA at 72 hours after carcinogen treatment, was chromatographically identical to a synthetic purine ring-opened derivative of 7-methylguanine and could be released from the isolated hepatic DNA by a specific glycosylase. The synthetic ring-opened adduct was characterized by mass and NMR spectroscopy as N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine and appears to exist in two rotameric forms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25317/1/0000762.pd