ANTHONY PEGG

..These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved...

..Several polymorphisms in the human alkyltransferase gene have been identified but the significance of these in terms of alkyltransferase action is currently unknown...

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..Such proteins occur naturally, and synthetic variants engineered to react specifically with derivatives of O(6)-benzylguanine are the basis of a valuable research technique for tagging proteins with specific reagents...

..The intricate mechanisms for regulation of mammalian S-adenosylmethionine decarboxylase activity and content are also described...

..Mutations in the human SMS lead to a rise in spermidine and reduction of spermine causing Snyder-Robinson syndrome, an X-linked recessive condition characterized by mental retardation, skeletal defects, hypotonia, and movement disorders...

..This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism...

..This review describes key factors that contribute to the regulation of ODC levels, which can occur at the levels of transcription, translation, and protein turnover...

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..DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication...

..These results indicate that the double mutant R101A/E152K-SSAT protein can be used to evaluate the importance of SSAT activity in response to exogenous polyamines or polyamine analogues...

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..J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase...

..These mice demonstrate for the first time that AZ suppresses tumor growth in an animal cancer model and provide a valuable model system to evaluate the role of ODC and polyamines in skin tumorigenesis...

..These results suggest mechanisms by which AGT may search for alkylated sites and interact with them to effect DNA repair...

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..Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)..

..Therefore these mice provide a model to study the in vivo co-operativity between high ODC activity and activation of other pathways leading to hypertrophy in the heart...

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..Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors which may have additional side effects...

..These transgenic mice thus provide a valuable model system in which to study the importance of polyamine levels in cardiac growth and electrophysiology in response to stress...

..The ability of the AGT proteins to form stable complexes with DNA was therefore examined by measuring the retardation of DNA during electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)..

..Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine...

..In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS...

..The development of other AGT inactivators which are still able to inactivate the resistant mutants may be necessary to maximize the potential of AGT inhibition for cancer chemotherapy...

..7Res SSAT cDNA. Expression of wtSSAT activity in C55.7Res cells restored sensitivity to bis(ethyl)polyamines. These results provided definitive evidence that SSAT activity is a critical target of the cytotoxic action of these analogues...

..This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor...

..This indicates that these compounds are substrates for the polyamine transport system, but that they are less effective than the natural polyamines in the feedback regulation of this system...

..These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group. Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC...

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..The hydrogen bond formed by the hydroxyl group from tyrosine-158 may also facilitate the reaction but the contribution from this interaction is relatively small...

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..Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N-methyl-N'-nitro-N-nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase...

..On the basis of these results, it was postulated that residue Cys-82 may be the proton donor of the decarboxylation reaction catalyzed by S-adenosylmethionine decarboxylase...

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..This interaction is prevented by mutations that modify the active site of AGT to exclude BG...

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..SSAT2 should be renamed 'thialysine N(epsilon)-acetyltransferase', and may regulate this pathway...

..These results show that spermine synthesis is needed for normal growth, viability, and fertility in male mice and that regulation of spermine synthase content is not required...

..We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates...

..These results indicate that this DNAzyme may be a useful tool to study the function of ODC and may have potential therapeutic uses...

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..Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation...

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..Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity...

..These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage...

..We hypothesize that modest binding cooperativity and high binding densities are adaptations that allow AGT to efficiently search for lesions in the context of chromatin remodeling and DNA replication...

..These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development...

..These results identify a pathway through which the alpha9 integrin subunit stimulates cell migration by localized polyamine catabolism and modulation of Kir channel function...

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..These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues...

..While MGMT may be important at the cellular level, mice deficient in MGMT are not significantly more susceptible to cyclophosphamide, acrolein or CAA. Thus, our data does not support targeting MGMT to improve oxazaphosphorine therapy...

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..Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases...

..These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein...

..These results suggest that pancreatic tumors, which are resistant to DNA alkylating agents, may be sensitized to such agents when pretreated with MGMT inactivators...

..These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide...

..These results suggest that the sequence specificity in the repair of O(6)mG is manifested in the methyl transfer not in the O(6)mG recognition step...

..In the presence of DNA, the DNA-binding function of AGT facilitates formation of DNA adducts. In the absence of DNA, the intermediate undergoes hydrolytic decomposition to form AGT-Cys(145)-SCH(2)CH(2)OH...

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..5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination...

..These findings will aid in designing novel AGT inhibitors that incorporate O(6)-alkylguanine adducts in oligodeoxyribonucleotide contexts...

..This may be of particular importance because such exposure may also lead to the formation of N-nitroso compounds that can act as alkylating agents...

..This is a novel type of post-translational modification causing ubiquitination...

..In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines...

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..These results suggest that activation of polyamine catabolism leading to increases in putrescine and N1-acetylspermidine may play a key role in chemically induced mouse skin neoplasia...

..Zinc also provides conformational stability to hAGT that may influence its regulation...

..The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles...

..These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair...

..The studies also provide a structural basis for the specificity of the spermidine synthase subclass of the aminopropyltransferase family...

..022 to 0.000016 s(-1). The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context...

..Because AGT is overexpressed in many tumor types, further investigations of the potential role of AGT-DNA cross-linking in the antitumor and mutagenic activity of antitumor nitrogen mustards are warranted...

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..The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif...

..A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs...

..The inactivation of these enzymes may mediate some of the antiproliferative actions of NO...

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..A high proportion of breast cancers induced SSAT in response to DENSpm, supporting the continued consideration of this class of agents for treatment of breast cancer...

..Both timing and dosing of selection regimens and the starting level of marking may all be important to the level of selective increase of in vivo marking achieved...

..These results demonstrate the crucial importance of ODC for the induction of BCCs and indicate that chemopreventive strategies directed at inhibiting this enzyme may be useful in reducing BCCs in human populations...

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..These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate beta-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver beta-glucuronidase to target tumor cells...

..Although agmatine is a known constituent of mammalian cells, it can be transported from the diet. Therefore L-ornithine decarboxylase remains the only established route for de novo putrescine biosynthesis in mammals...

..These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain...

..The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions...

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..Additionally, the presence of MR reflects a role for spermine in cognitive function, possibly by spermine's ability to function as an 'intrinsic gateway' molecule for inward rectifier K(+) channels...

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..These data establish a novel functional difference between the I143V/K178R protein and other hAGTs in the repair of a toxicologically relevant substrate, O(6)-pobG...

..AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein...

..The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn2+-dependent DNA repair activity may be relevant to the evolution and function of AGTs...

..abstract_text> ..

..Cells transfected with AGT, AGT mutants that may affect nuclear localization and with Ada-C will be examined for distribution of AGT and for protection from alkylating agents. ..

..They will also aid in the design of therapeutically useful polyamine antimetabolites and in the adoption of effective protocols using currently available agents. ..

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..A similar protocol to that used to obtain the BE-3-3-3 resistant cells will be used to isolate cells resistant to CHE-Spm which will then be used to study its mechanism of action. ..

..These experiments provide a means to reverse a well know source of resistance to alkylating agents that are used in such therapy. As such, they will increase the likelihood that these agents will produce therapeutic responses. ..

..The knowledge generated in this research will aid in the prevention and treatment of cancer. ..

..These experiments will aid in the understanding of the individual risks associated with exposure to alkylating agents and an important class of environmental carcinogens. ..

..5) To characterize and evaluate the biological significance of the differences in hAGT activity imparted by different polymorphic forms of the protein. ..

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..The enzyme responsible for the removal of O6-methylguanine from rat liver has been partially purified and the properties of this enzyme are currently under investigation...

..8-Azido derivatives of decarboxylated AdoMet and MTA have been synthesized and will be used as photoactivatable probes for spermidine synthase, spermine synthase and MTA phosphorylase...