One of the most common microsatellites in eukaryotes consists of tandem arrays usually 15-50 base pairs (bp) in length of the dinucleotide GT. We examined the rates of instability for poly GT tracts ...of 15, 33, 51, 99 and 105 bp in wild-type and mismatch repair-deficient strains of Saccharomyces cerevisiae. Rates of instability increased more than two orders of magnitude as tracts increased in size from 15 to 99 bp in both wild-type and msh2 strains. The types of alterations observed in long and short tracts in wild-type strains were different in two ways. First, tracts > or = 51 bp had significantly more large deletions than tracts < or = 33 bp. Second, for the 99- and 105-bp tracts, almost all events involving single repeats were additions; for the smaller tracts, both additions and deletions of single repeats were common.
CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As ...activation of CPT-11 (irinotecan-7-ethyl-10-4-(1-piperidino)-1-piperidinocarbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC(50) value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.
A sequence (sn) of integers is good for the mean ergodic theorem if for each invertible measure-preserving system (X,ℬ,μ,T) and any bounded measurable function f, the averages (1/N)∑ Nn=1f(Tsnx) ...converge in the L2(μ) norm. We construct a sequence (sn) which is good for the mean ergodic theorem but such that the sequence (s2n) is not. Furthermore, we show that for any set of bad exponents B, there is a sequence (sn) where (skn) is good for the mean ergodic theorem exactly when k is not in B. We then extend this result to multiple ergodic averages of the form (1/N)∑ Nn=1f1(Tsnx)f2(T2snx)⋯fℓ(Tℓsnx). We also prove a similar result for pointwise convergence of single ergodic averages.
Irinotecan, 7-ethyl-10-4-(1-piperidino)-1-piperidinocarbonyloxycamptothecin (CPT-11) is activated by carboxylesterases (CE) to yield the potent topoisomerase I inhibitor, SN-38. We have demonstrated ...previously that a rabbit liver CE is approximately 100-1000-fold more efficient at drug activation than a highly homologous human CE. In an attempt to use rabbit CE expression in combination with CPT-11 for gene therapy approaches for the treatment of cancer, we have developed an adenoviral vector expressing this intracellular CE. After transduction, this virus produces very high levels of CE activity in a panel of human tumor cell lines and results in marked sensitization to CPT-11 of all of the transduced cells. Reductions in IC(50) values for this drug ranged from 11-127-fold. Additionally, comparison with an adenovirus expressing a secreted form of the rabbit CE indicated that a collateral effect could be achieved with reductions in the IC(50) values ranging from 4-19-fold. These data suggest that the described reagents may be suitable for use in vivo in a viral-directed enzyme prodrug therapy approach using CPT-11.
We prove pointwise convergence, as N → ∞, for the multiple ergodic averages $(1/\mathrm{N})\sum _{\mathrm{n}=1}^{\mathrm{N}}\mathrm{f}\left({\mathrm{T}}^{\mathrm{n}}\mathrm{x}\right)\cdot ...\mathrm{g}\left({\mathrm{S}}^{{\mathrm{a}}_{\mathrm{n}}}\mathrm{x}\right)$, where T and S are commuting measure preserving transformations, and an is a random version of the sequence nc for some appropriate c > 1. We also prove similar mean convergence results for averages of the form $(1/\mathrm{N})\sum _{\mathrm{n}=1}^{\mathrm{N}}\mathrm{f}\left({\mathrm{T}}^{{\mathrm{a}}_{\mathrm{n}}}\mathrm{x}\right)\cdot \mathrm{g}\left({\mathrm{S}}^{{\mathrm{a}}_{\mathrm{n}}}\mathrm{x}\right)$, as well as pointwise results when T and S are powers of the same transformations. The deterministic versions of these results, where one replaces an with nc, remain open, and we hope that our method will indicate a fruitful way to approach these problems as well.
The camptothecin prodrug CPT-11 (irinotecan, 7-ethyl-10-4-(1-piperidino)-1-piperidinocarbonyloxycamptothecin) is converted by esterases to yield the potent topoisomerase I poison SN-38 ...(7-ethyl-10-hydroxycamptothecin). Recently, a mouse strain (Es1(e)) has been identified that demonstrates reduced plasma esterase activity, and we have monitored the ability of plasma from these mice to metabolize CPT-11. Total plasma esterase activity was reduced 3-fold in Esl(e)mice in comparison to control mice, and this resulted in a 200-fold reduction in SN-38 production after incubation with CPT-11 in vitro. In addition, pharmacokinetic studies of CPT-11 and SN-38 in these animals demonstrated approximately 5-fold less conversion to SN-38. However, extracts derived from tissues from Es1(e) animals revealed total esterase activities similar to those of control mice, and these extracts metabolized CPT-11 with equal efficiency. Northern analysis of RNA isolated from organs indicated that the liver was the primary source of Es-1 gene expression and that very low levels of Es-1 RNA were present in Es1(e) mice. These results suggest that the reduced levels of Es-1 esterase present in Es1(e) mice are due to down-regulation of gene transcription, and that this plasma esterase is responsible for the majority of CPT-11 metabolism in mice.
Simple repetitive DNA sequences in the eukaryotic genome frequently alter in length. In wild-type strains, we find that transcription through a repetitive poly GT tract destabilizes the tract four- ...to ninefold. In mismatch repair-deficient yeast strains, simple repeats are very unstable. High levels of transcription in such strains destabilize repetitive tracts an additional two- to threefold
Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, ...effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-4-(1-piperidino)-1-piperidinocarbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%.
Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of ...selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K i values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K i (r 2 > 0.91), with cross-validation coefficients (q 2) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.
Let a(x) be a real function with a regular growth as x → ∞ . The precise technical assumption is that a(x) belongs to a Hardy field. We establish sufficient growth conditions on a(x) so that the ...sequence (a(n))n=1∞ is a good averaging sequence in L2 for the pointwise ergodic theorem. A sequence (an) of positive integers is a good averaging sequence in L2 for the pointwise ergodic theorem if in any dynamical system (Ω , Σ , m, T) for f ∈ L2(Ω ) the averages 1/X∑n≤ Xf(Tanω ) converge for almost every ω ∈ Ω . Our result implies that sequences like (nδ), where δ > 1 and not an integer, (n log n), and (n2/log n) are good averaging sequences for L2. In fact, all the sequences we examine will turn out to be good averaging for Lp, p > 1; and even for L log L. We will also establish necessary and sufficient growth conditions on a(x) so that the sequence (a(n)) is good averaging for mean convergence. Note that for some a(x) (e.g., a(x) = log2 x), (a(n)) may be good for mean convergence without being good for pointwise convergence.
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