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61.
D Schuler M Otteneder P Sagelsdorff E Eder RC Gupta WK Lutz 《Canadian Metallurgical Quarterly》1997,18(12):2367-2371
8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P-postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction. 相似文献
62.
ME Dolan SK Roy BJ Garbiras P Helft P Paras MY Chae RC Moschel AE Pegg 《Canadian Metallurgical Quarterly》1998,55(10):1701-1709
To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types. 相似文献
63.
SI Becker R Wang RC Hedstrom JC Aguiar TR Jones SL Hoffman MJ Gardner 《Canadian Metallurgical Quarterly》1998,66(7):3457-3461
Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund's adjuvant. 相似文献
64.
C Robinson SJ Brookes J Kirkham WA Bonass RC Shore 《Canadian Metallurgical Quarterly》1996,10(2):173-9; discussion 179-80
Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins. 相似文献
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66.
The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family 总被引:2,自引:0,他引:2
A physiological role for paraoxonase (PON1) is still uncertain, but it catalyzes the hydrolysis of toxic organophosphates. Evidence that the human genome contains two PON1-like genes, designated PON2 and PON3, is presented here. Human PON1 and PON2 each have nine exons, and the exon/intron junctions occur at equivalent positions. PON1 and PON2 genes are both on chromosome 7 in human and on chromosome 6 in the mouse. Turkey and chicken, like most birds, lack paraoxonase activity and are very susceptible to organophosphates. However, they have a PON-like gene with approximately 70% identity with human PON1, PON2, and PON3. Another unexpected finding is that the deduced amino acid sequences of PON2 in human, mouse, dog, turkey, and chicken and of human PON3 are all missing the amino acid residue 105, which is lysine in human PON1. The expanded number of PON genes will have important implications for future experiments designed to discover the individual functions, catalytic properties, and physiological roles of the paraoxonases. 相似文献
67.
ID Kurzman EG MacEwen RC Rosenthal LE Fox ET Keller SC Helfand DM Vail RR Dubielzig BR Madewell CO Rodriguez 《Canadian Metallurgical Quarterly》1995,1(12):1595-1601
Two randomized, double-blind clinical trials in dogs with spontaneous appendicular osteosarcoma treated with combination chemoimmunotherapy are reported. In both trials, dogs without overt metastasis underwent complete amputation of the affected limb. In trial 1, 40 dogs were treated with cisplatin chemotherapy [(CDDP), 70 mg/m2 i.v. every 28 days x 4]. Following CDDP, dogs without evidence of overt metastasis (n = 25) were randomized to receive liposome-encapsulated muramyl tripeptide phosphatidylethanolamine ](L-MTP-PE), 2 mg/m2 i.v.) or placebo liposomes (lipid equivalent) twice weekly for 8 weeks. Of 14 dogs in the placebo group, 13 (93%) died of metastasis; the median survival time was 9.8 months. Of 11 dogs in the L-MTP-PE group, 8 (73%) developed metastasis; the median survival time was 14.4 months, which was significantly longer than that of the placebo group (P < 0.01). In trial 2, 64 dogs received CDDP (70 mg/m2 i.v. every 21 days x 4) and were randomized to concurrently receive L-MTP-PE (2 mg/m2 i.v.) twice or once weekly, or placebo liposomes once weekly for 8 weeks. Median survival times were 10.3, 10.5, and 7.6 months, respectively. There were no significant differences among the three treatment groups in trial 2. Survival times for dogs receiving L-MTP-PE in trial 1 were significantly longer than those for dogs in trial 2 that received four doses of CDDP concurrently with twice weekly L-MTP-PE (P < 0. 04). The results of the first trial confirm our previous observation that L-MTP-PE has antimetastatic activity in dogs with osteosarcoma when given following amputation. The results of the second trial demonstrate that there is no survival advantage of administering L-MTP-PE concurrently with CDDP. 相似文献
68.
SH Kaufmann D Peereboom CA Buckwalter PA Svingen LB Grochow RC Donehower EK Rowinsky 《Canadian Metallurgical Quarterly》1996,88(11):734-741
BACKGROUND: Topotecan (TPT) is a topoisomerase I poison that exhibits antineoplastic activity. Analysis of the cytotoxic effects of combinations of TPT and other anticancer agents has been limited. PURPOSE: We assessed the cytotoxic effects produced by combinations of TPT and other antineoplastic agents in experiments involving multiple human cancer cell lines of diverse histologic origins. METHODS: The cytotoxic effects of various antimetabolites (fluorouracil, methotrexate, or cytarabine), antimicrotubule agents (vincristine or paclitaxel [Taxol]), DNA alkylating agents (melphalan, bis[chloroethyl]nitrosourea [BCNU], or 4-hydroperoxycyclophosphamide [4HC]), and a DNA-platinating agent (cisplatin), alone and in combination with TPT, were measured in clonogenic (i.e., colony-forming) assays. HCT8 ileocecal adenocarcinoma, A549 non-small-cell lung carcinoma, NCI-H82ras(H) lung cancer, T98G glioblastoma, and MCF-7 breast cancer cell lines were used in these assays. The data were analyzed by the median effect method, primarily under the assumption that drug mechanisms of action were mutually nonexclusive (i.e., completely independent of one another). For each level of cytotoxicity (ranging from 5% to 95%), a drug combination index (CI) was calculated. A CI less than 1 indicated synergy (i.e., the effect of the combination was greater than that expected from the additive effects of the component agents), a CI equal to 1 indicated additivity, and a CI greater than 1 indicated antagonism (the effect of the combination was less than that expected from the additive effects of the component agents). RESULTS: When the mechanisms of drug action were assumed to be mutually nonexclusive, virtually all CIs for combinations of TPT and either antimetabolites or antimicrotubule agents revealed cytotoxic effects that were less than additive. The CIs calculated at low-to-intermediate levels of cytotoxicity for combinations of TPT and the DNA alkylating agents melphalan, BCNU, and 4HC also showed drug effects that were less than additive; in most cases, however, nearly additive or even synergistic effects were observed with these same drug combinations at high levels of cytotoxicity (i.e., at > or = 90% inhibition of colony formation). Results obtained with combinations of TPT and cisplatin varied according to the cell line examined. With A549 cells, less than additive effects were seen at low-to-intermediate levels of cytotoxicity, and more than additive effects were seen at high levels of cytotoxicity. With NCI-H82ras(H) cells, synergy was observed over most of the cytotoxicity range. CONCLUSIONS AND IMPLICATIONS: TPT cytotoxicity appears to be enhanced more by combination with certain DNA-damaging agents than by combination with antimetabolites or antimicrotubule agents. Interactions between TPT and other drugs can vary depending on the cell type examined. Further investigation is required to determine the basis of the observed effects and to determine whether these in vitro findings are predictive of results obtained in vivo. 相似文献
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70.