Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N 6 -ethenoadenine (A) and 3,N 4 -ethenocytosine (C) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises A. The present work is directed toward identifying and purifying the human glycosylase activity releasing C. HeLa cells were subjected to multiple steps of column chromatography, including two C-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS͞polyacrylamide gel showed that the C activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G͞T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having C-DNA glycosylase activity. In addition, oligonucleotides containing C⅐G or G͞T(U), could compete for C protein binding, further indicating that the C-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for C also was observed in a recombinant G͞T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.The four etheno adducts of DNA and RNA have been of considerable interest to organic chemists and physical scientists due to their physical, chemical and spectroscopic properties which had broad applications in protein-nucleic acid interactions and DNA structure [reviewed by Leonard (1, 2) and refs therein]. These adducts became of major interest when they were found to be formed by a variety of environmental agents, as well as produced endogenously (3-7). Mutagenesis studies have shown a wide range of mutagenic frequency depending on the type of the adduct, type of mutation and the system used for detection and quantitation (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).For more than one decade, this laboratory has focused on studies on the repair (20-26) and replication͞transcription (8-12) of etheno derivatives of dA, dC, and dG. The differing structures of the etheno adducts and their effect on base pairing and base stacking (27-31) influences both repair and replication. Much of the data has been obtained by using prokaryotic systems, which are not always identical to those now found in the more widely used mammalian systems. It was established earlier in repair studies, by using a human system, that all four etheno adducts were released by HeLa cell-free extracts, indicating that they are substrates for DNA glycosylases (24). After partial purification from HeLa cells, 3,N 4 -ethenocytosine (C) repair activity was found to be separate from 1,N 6 -ethenoadenine (A) repair activity (25), which is a function of alkylpurine-DNA-N-glycosylase (APNG) (22). A knockout mouse lacking APNG was then used as a genetic approach to verify these in vitro data (26). Under these c...

There is currently no "gold standard" test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (n ‫؍‬ 195), healthy controls (n ‫؍‬ 400), and patients with other parasitic diseases (n ‫؍‬ 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA IA proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA IA proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania. These data suggest the need for field validation studies of TESA-and TESA IA -based assays in regions where Chagas' disease is endemic.

Mixed infestation of nymphs and adults of Rhodnius prolixus Stal, 1859 and Panstrongylus geniculatus Latreille, 1811 was detected in 3 (15%) of 20 dwellings in El Guamito, an endemic focus of Chagas disease in Lara State, Venezuela. In one of the houses, both species were positive for Trypanosoma cruzi: 14.3% (R. prolixus) and 20% (P. geniculatus ). The overall infection rate in 143 of 352 R. prolixus was 16.1%. Parasites isolated from R. prolixus were identified as T. cruzi I by random amplified polymorphic DNA analysis. Dot-enzyme-linked immunosorbent assays of 36 R. prolixus showed that 58.3% of the R. prolixus had fed on humans. The gut contents of one fifth-instar nymph of P. geniculatus that was positive for T. cruzi also reacted with anti-human serum. A questionnaire was used to gather data on the demographic and socioeconomic characteristics of the population. An indirect immunofluorescent test, an indirect hemaglutination test, and an ELISA were used to detect the presence of antibodies against T. cruzi in 84 of 86 inhabitants and in 15.5% of people more than 20 years old. The relative risk (RR) of infection was greater in men than in women (RR = 1.61, 95% confidence interval = 0.54-4.80). Of the people more than 15 years old, 36.6% had no formal education. All respondents recognized triatomine bugs, but they did not relate them to Chagas disease transmission. A total of 85.7% of the houses were "ranchos" suitable for the colonization of triatomine bugs. The possible domiciliation of P. geniculatus and the implications of competition with R. prolixus for resources are discussed. Since there is no clear separation of food sources, abiotic factors such as microclimatic variation within houses may be critical to predict the outcome of the process of competition and potential domestication of this generally sylvatic species.

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