Magnetic resonance imaging and image analysis of post - radiation changes of bone marrow in patients with skeletal metastases.

PURPOSE: To evaluate the post-radiation lesions of the bone marrow with magnetic resonance imaging (MRI) and image analysis in patients with bone metastases undergoing radiation therapy (RT). METHODS: Thirty-five patients with bone metastases were studied from June 2008 to December 2010. All patients had osseous metastases from various primary malignancies and underwent palliative RT. MRI was performed in a Philips Gyroscan Intera 1T scanner at the beginning of RT and 12-18 days later. T1-TSE, T2-TSE and short tau inversion recovery (STIR) sequences were used. All images obtained were evaluated for early post-radiation lesions. Additionally, 1st and 2nd order textural features were extracted from these images and were introduced into a probabilistic neural network (PNN) classifier in order to create an automated classification system for those lesions. RESULTS: Changes of signal intensity in T1-TSE, T2-TSE and STIR sequences were evaluated for the presence of edema, fatty conversion of the bone marrow or areas of hemorrhage within the limits of the irradiated area. The automated classification system showed positive results in correctly discriminating the post-radiation lesions that MRI revealed. The overall classification accuracy for discriminating between pre-radiation and post-radiation lesions was 93.2%. Furthermore, the overall classification accuracy for discriminating between post-radiation lesions was 86.67%. CONCLUSION: It seems that MRI can evaluate the degree of early therapy-induced bone marrow lesions observed during the first 18 days from the beginning of RT. The proposed neural network-based classification system might be used as an assisting tool for the characterization of these lesions.

Thyroid cancer in Greece. A tertiary center experience.

PURPOSE: to present the frequency and distribution of histological subtypes of primary thyroid carcinoma cases diagnosed at our Department. METHODS: we retrospectively analyzed the records of all patients with primary thyroid carcinoma admitted to our Department from January 01, 2002 until December 31, 2008. RESULTS: of 1,607 patients with primary thyroid carcinoma 1510 (94%) suffered from differentiated thyroid carcinoma (DTC), 71 (4%) from medullary thyroid carcinoma (MTC), 10 (< 1%) from anaplastic thyroid carcinoma (ATC), 9 (<1%) from primary thyroid non Hodgkin's lymphoma, 1 (< 1%) from primary thyroid Hodgkin's lymphoma, 1 (< 1%) from squamous cell carcinoma of the thyroid, and 6 patients (< 1%) suffered from carcinoma of ectopic thyroid tissue (1 malignant struma ovarii, 2 carcinoma in thyroglossal duct remnants, and 3 patients presented with thyroid tissue carcinoma in cervical lymph nodes). CONCLUSION: differentiated thyroid carcinoma is by far the most common primary malignancy of the thyroid gland. Papillary thyroid carcinomas constitute the vast majority of these neoplasms. Larger studies are necessary to better understand and evaluate the clinical characteristics, behavior and prevalence of thyroid cancer in our country.

Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Benzo[a]pyrene-enhanced mutagenesis by man-made mineral fibres in the lung of lamda-lacI transgenic rats.

In an attempt to examine the interaction of man-made mineral fibres with benzo[a]pyrene (B[a]P), homozygous X-lacI transgenic F344 rats were intratracheally treated with rock (stone) wool RWI and glass wool MMVF 10 fibres together with B[a]P. To analyze the induction of gene mutations by fibres and B[a]P in lung, single doses of 1 and 2 mg fibres/animal or multiple doses of 2 mg fibres/animal were administered weekly on 4 consecutive weeks (total dose 8 mg/animal). B[a]P (10 mg/animal) was administered either simultaneously with fibres (for single dose treatment with fibres) or together with the last fiber treatment (for multiple dose treatment with fibres). Animals were scarified 4 weeks after the last treatment. Benzo[a]pyrene administered simultaneously with RW1 fibres exhibited a strong synergistic effect on mutagenicity, the observed mutant frequency (MF) being more than three-fold higher than the net sum of the MF induced after separate administration of both agents. Our data suggest that DNA adducts induced by simultaneous B[a]P and fiber treatment lead to a strong increase in mutatant frequencies.

Mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

Benzo[a]pyrene-enhanced mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to lambdalacI transgenic rats, together with 1, 2 or 4 x 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis.

Impact of phase I or phase II enzyme polymorphisms on lymphocyte DNA adducts in subjects exposed to urban air pollution and environmental tobacco smoke.

Little is known about the impact of genetic variation on the genetic damage induced by urban air pollution or environmental tobacco smoke (ETS) in exposed populations. The levels of bulky DNA adducts ( 32P-postlabelling, nuclease P1 enrichment) and chromosomal aberrations were measured in lymphocytes of 194 non-smoking students living in the city of Athens, and the rural region of Halkida, Greece. In these individuals personal exposure to PAH was also measured. Furthermore, genetic polymorphisms were examined in cytochromes P450 1A1, 1B1, in the GSTM1, GSTP1 and GSTT1 as well as in microsomal epoxy hydrolase (EPHX) genes. Subjects with the CYP1*2A mutant genotype also suffering significant ETS exposure tended to exhibit higher adduct levels and % aberrant cells. In contrast, CYP1B1 polymorphisms seemed to have an impact on the DNA adduct levels only among individual with negligible ETS exposure. Subjects carrying both the CYP1*2A mutant genotype and the GSTM1 null genotype tended to have higher DNA adduct levels. A similar effect was also observed with the combined CYP1A1*2A/GSTP1 (Ile/Val) and the CYP1A1*2A/mEH "slow" polymorphisms. In both cases, the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise restriction of the observations to subjects characterised by (a) GSTP1 mutant, (b) GSTM1 null, (c) mEH "slow" (His139His) genotypes and (d) ETS exposure resulted in a significant trend of increasing DNA adduct levels only among individuals with at least one CYP1A1*2A mutated allele, illustrating the importance and complexity of gene-exposure and gene-gene interactions in determining the level of genotoxic damage on an individual levels.

Using handheld devices for real-time wireless teleconsultation.

Recent advances in the hardware of handheld devices, opened up the way for newer applications in the healthcare sector, and more specifically, in the teleconsultation field. Out of these devices, this paper focuses on the services that personal digital assistants and smartphones can provide to improve the speed, quality and ease of delivering a medical opinion from a distance and laying the ground for an all-wireless hospital. In that manner, PDAs were used to wirelessly support the viewing of digital imaging and communication in medicine (DICOM) images and to allow for mobile videoconferencing while within the hospital. Smartphones were also used to carry still images, multiframes and live video outside the hospital. Both of these applications aimed at increasing the mobility of the consultant while improving the healthcare service.

Biomarkers of genotoxicity of urban air pollution. Overview and descriptive data from a molecular epidemiology study on populations exposed to moderate-to-low levels of polycyclic aromatic hydrocarbons: the AULIS project.

Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

Biomarkers of genotoxicity of air pollution (the AULIS project): bulky DNA adducts in subjects with moderate to low exposures to airborne polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke and other parameters.

The levels of bulky DNA adducts were measured by (32)P-post-labelling in lymphocytes of 194 non-smoking students living in the city of Athens and the region of Halkida, Greece, once in the winter and again in the following summer. Personal exposures to particulate-bound polycyclic aromatic hydrocarbons (PAH) were significantly higher in Athens subjects during both seasons. There was hardly any diagonal radioactive zone in the pattern of DNA adducts observed. Highest adduct levels were observed in a sub-group of subjects living in or near the Halkida Institute campus, which was located in rural surroundings with a minimal burden of urban air pollution. The remaining Halkida subjects had intermediate levels, while Athens subjects showed the lowest levels. This trend, which was observed over both monitoring seasons, consistently paralleled the variation in three markers of exposure to environmental tobacco smoke (ETS), namely (i) declared times of exposure to ETS during the 24 h prior to blood donation, (ii) plasma cotinine levels and (iii) chrysene/benzo[g,h,i]perylene ratios in the profile of personal PAH exposure. Furthermore, among the Halkida campus area subjects (but not the remaining subjects) positive correlations were observed between DNA adducts and (i) measured personal exposures to chrysene or benzo[a]pyrene, (ii) time of declared ETS exposure and (iii) chrysene/benzo[g,h,i] perylene ratios. These correlations suggest that, for a group suffering minimal exposure to urban air pollution, exposure to ETS was a significant determinant of the observed DNA damage. Gender had a consistent and significant effect on adduct levels (males having higher levels), which remained significant even after multiple regression analysis. Habitual consumption of roasted meat was significantly associated with an enhancement of adduct levels and the effect was strengthened when only individuals unexposed to ETS were taken into consideration. No significant effects were observed for other dietary parameters or factors reflecting exposure to air pollution.

Personal exposures to PM(2.5) and polycyclic aromatic hydrocarbons and their relationship to environmental tobacco smoke at two locations in Greece.

In the context of a large-scale molecular epidemiology study of biomarkers of genotoxicity of air pollution, 24-h mean personal exposures to airborne PM(2.5) (particulate matter <2.5 microm) and associated polycyclic aromatic hydrocarbon (PAHs) were measured in 194 non-smoking technical institute students living in the city of Athens, Greece (an area with moderately high levels of air pollution) and the nearby small town of Halkida anticipated to have lower pollution levels. Extensive information relevant to the assessment of long-term and recent exposure to PAH was obtained from questionnaires as well as a time-location-activity diary (TLAD) which was kept by all subjects during a 4-day observation period. During the last 24 h of this period, subjects underwent personal exposure monitoring for PM(2.5) and PAH, while a sample of blood was donated at the end of this period. All subjects were monitored in this way twice; once during a winter season (October-February) and once during the following summer season (June-September). Nine subjects with plasma cotinine levels above 20 ng/ml were considered as unreported smokers and excluded from the study. Winter PM(2.5) exposures were lower in Athens (geometric mean 39.7 microg/m(3)) than Halkida (geometric mean 56.2 microg/m(3)) (P<0.001), while there was no significant location difference during the summer (Athens: geometric mean 32.3 microg/m(3), Halkida: geometric mean 32.9 microg/m(3); P=0.79). On the other hand, PAH exposures (sum of the eight carcinogenic PAHs) were significantly higher in Athens than in Halkida during the winter (Athens: geometric mean 8.26 ng/m(3), Halkida: geometric mean 5.80 ng/m(3); P<0.001) as well as during the summer (Athens: geometric mean 4.44 ng/m(3), Halkida: geometric mean 1.48 ng/m(3); P<0.001). There was a significant difference in the profile of the PAH exposures at the two locations, the proportion of lighter PAH (benzo[a]anthracene, chrysene [CHRYS], benzo[k]fluoranthene, and benzo[b]fluoranthene) being higher, and that of heavier PAH (benzo[ghi]perylene [BPer] and indeno[1,2,3,cd]pyrene) lower, in Halkida than in Athens, regardless of season. This difference appeared to be related to individual exposure to environmental tobacco smoke (ETS), as indicated by (a) the correlation at the individual level between the CHRYS/BPer ratio and declared time of recent exposure to ETS as well as plasma cotinine levels, especially during the winter; (b) the parallel variation of the mean levels of all three markers (declared ETS exposure, cotinine levels, CHRYS/BPer ratio) among three subgroups of subjects (Athens subjects who had lowest levels of all three markers; Halkida subjects other than those living in the institute campus area; and Halkida subjects living in the institute campus area who had the highest levels of all three markers). This demonstrates that ETS can have a distinctive effect on the PAH exposure profile of subjects exposed to relatively low levels of urban air pollution.

Ubiquitous presence of O6-methylguanine in human peripheral and cord blood DNA.

O6-Methylguanine (O6-meG) is a powerful premutagenic lesion that can arise from exposure to methylating agents. Although it has been reported to occur in human DNA, no systematic epidemiological analysis of its occurrence in populations suffering general environmental exposure is available. We report here results from a study of the presence of O6-meG in maternal and cord blood leukocyte DNA of women not knowingly exposed to methylating agents. Using a modification of an already existing method capable of detecting the lesion at levels as low as 16 nmol/molG, the adduct was detected in 31 of 36 maternal and 30 of 36 cord samples, at levels ranging up to 192 nmol/molG. Adduct levels in maternal blood DNA were significantly higher than those in cord blood DNA (P < 0.05), and there was a strong correlation between adduct levels in the two tissues (P < 0.001). In bivariate analysis, no significant association of adduct levels in either tissue and residence air pollution, active and passive smoking status, or eating habits was found. However, intake of fruits/vegetables and of vitamin supplements showed nonstatistically significant trends toward being associated with lower adduct levels in both maternal and cord blood DNA. The same trend was observed after multivariate analysis where all the above variables were controlled for. These findings indicate that premutagenic methylation DNA damage is commonplace in individuals not known to have suffered excessive exposure to environmental methylating agents or their precursors and are compatible with an endogenous origin of this damage, possibly associated with endogenous nitrosation processes.

Molecular epidemiological approaches to the study of the genotoxic effects of urban air pollution.

Direct epidemiological observations suggest that exposure to high levels of urban air pollution may result in increased risk of lung cancer, sufficient to account for a few (approximately 1-3) percent of total lung cancer incidence. Extrapolation from occupational exposure and risk data suggests that among potential carcinogens present in polluted urban air, polycyclic aromatic hydrocarbons (PAHs) may make a major contribution to air pollution-associated lung cancer risks. The use of biomarkers of genotoxocity in large-scale population studies may help to reduce the uncertainty involved in the assessment of such risks, especially those associated with relatively low pollution levels such as nowadays found in many Western cities. Increases in biomarkers of exposure to urban air PAHs as well as biomarkers of early effects have been detected in situations of relatively high levels of air pollution (e. g., ambient PAH concentrations of the order of a few tens of micrograms per cubic meter). Evidence has also been found about the modulation genetic damage accumulation in different individuals by polymorphisms in genes involved in the activation or detoxification of PAHs, especially of polymorphisms GSTM1 and CYP1A1 genes. However, the inconsistencies in the currently reported effects of genetic polymorphisms suggest that additional factors may also be important in the modulation of individual susceptibility to the accumulation of PAH-derived genetic damage. Biomarkers studies in populations exposed to relatively low ambient PAH concentrations (below 20 microg/m(3)) have not demonstrated clear dose-related effects (e.g., on DNA adduct levels), possibly because of the existence of multiple sources and routes of human exposure to PAHs in addition to inhalation of urban air (including, for example, home heating, environmental tobacco smoke and diet), and the consequent difficulty of adequately and specifically assessing atmospheric air-related exposure. This makes it imperative that molecular epidemiology studies be designed in such a way as to allow adequate assessment of exposure to urban air PAHs at the individual level and over short-, medium- and long-term time periods which correspond to the expression times of different biomarkers.

DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental and clinical significance.

DNA adducts are covalent complexes formed between genotoxic carcinogens and DNA bases, and constitute a critical early intermediate on the pathway of chemical carcinogenesis. Their accumulation in different tissues reflects the amount of activated carcinogen reaching DNA, and can therefore serve as an index of the biologically relevant dose reaching the target tissues or cells. Methylating agents are of interest in view of their occurrence in the environment and their use as cytotoxic drugs in cancer chemotherapy. Current evidence indicates that O6-methylguanine plays a particularly important role in the mutagenic, carcinogenic, and cytotoxic activities of methylating agents. O6-Methylguanine is repaired efficiently by the enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Lack of this enzyme results in excessive accumulation of O6-methylguanine and recent evidence suggests that significant quantitative effects on adduct accumulation may be linked to conditions of very low AGT levels. This would be important from the point of view of clinical practice, since modulation of AGT is under investigation as a means of enhancing the therapeutic efficacy of clinical agents acting via the production of O6-methylguanine and related adducts, such as, for example, procarbazine, dacarbazine, and some nitrosoureas. The measurement of O6-methylguanine in human DNA has been employed as a tool to investigate the role of environmental methylating agents in human carcinogenesis. While the nature and origin of the methylating agents responsible for these adducts is currently unknown, recent studies in patas monkeys have shown that N-nitrosodimethylamine, a methylating carcinogen to which human exposure is well documented, is capable of efficiently generating O6-methylguanine in most tissues, including fetal tissues. Furthermore, it has been found that this damage is substantially enhanced by the coadministration of ethyl alcohol which acts by inhibiting the liver first-pass metabolism of the carcinogen, an observation which supports the hypothesis that alcohol consumption may act as a risk factor in human carcinogenesis by augmenting the action of nitrosamines.

Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras.

Mammary and skin tumors induced in rodents by N-methyl-N-nitrosourea treatment have a G:C to A:T transition mutation in codon 12 of H-ras, probably resulting from alkylation of O6 of guanine by the carcinogen. This codon contains two guanines (5'-GGA-3'), but mutations are observed only in the central base pair of this codon. The same selectivity for mutations of -GGA-sequences has also been observed in Escherichia coli. It is known that the central G in the sequence GGA is a preferred site for alkylation, but the magnitude of chemical selectivity is insufficient to provide a complete explanation for the biological observation which is still unexplained. We have measured accurate rates of repair by the E. coli and gene O6-alkylguanine-DNA-alkyltransferase of an O6-methylguanine in various positions in chemically synthesized 15-base pair DNA duplexes having the H-ras sequence. The rate of repair varied 25-fold, depending on the sequence flanking the methylguanine. An O6-methylguanine in position 2 of codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation for the selective mutation of this position. Using an antibody to probe the accessibility of the O6-methyldeoxyguanosine, it was shown that the rate of repair is a reflection of the conformation of the sequence containing the alkylated base, because the avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the methylguanine, with the most rapidly repaired O6-methylguanines being those most easily bound by the antibody.

Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.