Interleukin-27 and interleukin-23 in patients with systemic lupus erythematosus: possible role in lupus nephritis.

OBJECTIVES: To analyse the concentration of interleukin (IL)-27 and IL-23 in serum and urine of patients with systemic lupus erythematosus (SLE) compared with healthy controls (HC). METHOD: An enzyme-linked immunosorbent assay (ELISA) was used to analyse the serum and urine concentration of IL-27 and IL-23 from 50 patients with lupus nephritis (LN), 55 patients without LN, and 30 HC. The correlations between the levels of IL-27, IL-23, and disease activity, clinical parameters in SLE patients were analysed. RESULTS: The levels of IL-27 and IL-23 increased significantly in the serum and urine of SLE patients with and without LN compared with HC. Moreover, urine levels of IL-27 and IL-23 were correlated with the renal SLE Disease Activity Index (rSLEDAI) score and 24-h urinary protein levels. After 6 months of immunosuppressive treatment, urine IL-27 expression rose significantly in SLE patients with LN. CONCLUSIONS: IL-27 and IL-23 may be involved in the pathogenesis of LN.

[Prevalence of human soil-borne nematode infections in Yunnan Province: a cross-sectional study in 2015].

OBJECTIVE: To investigate the current prevalence of human soil-borne nematode infections in Yunnan province, so as to provide the scientific evidence for formulating the soil-borne nematodiasis control strategy in the province. METHODS: In 2015, a total of 20 survey sites were sampled in 10 counties (cities) of Yunnan Province using the stratified cluster random sampling method. Stool samples were collected from all local permanent residents at ages of one year and older in each survey site, and the soil-borne nematode eggs were identified using the modified Kato-Katz technique and the egg number was counted. In addition, the hookworm species was identified using the filter-paperculture method, and Enterobius vermicularis eggs were detected using the cellophane tape method in children at ages of 3 to 6 years. RESULTS: A total of 5 067 residents received stool examinations, and 950 residents were detected with soil-borne nematode infections, with an overall prevalence rate of 18.75%. The prevalence of Ascaris lumbricoides, Trichuris trichura and hookworm was 7.52%, 8.47% and 9.02%, respectively. Among 446 children detected using the cellophane tape method, 5 children were detected with E. vermicularis infections. Among the 160 residents with hookworm infections, there were 139 residents with Necator americanus infections (86.88%), 16 with A. duodenale infections (10.00%) and 5 with mixed infections (3.12%). Mild A. lumbricoides (67.98%, 259/381), T. trichura (88.58%, 380/429) and hookworm infections (94.53%, 432/457) were predominant. Among the four ecological zones, the highest prevalence of human soilborne nematode infections was found in the East Tibet-South Sichuan Ecological Zone (31.79%), and among the 10 survey counties (cities), the greatest prevalence was seen in Gongshan Derung and Nu Autonomous County (50.13%), while the lowest prevalence was found in Ninglang Yi Autonomous County (0.40%). The prevalence of human soil-borne nematode infections was 5.67% (43/759), 26.67% (610/2 287) and 14.70% (297/2 021) in high-, moderate- and low-economic-level regions, respectively. There were no significant differences in the prevalence of human soil-borne nematode infections in terms of ecological regions, survey counties (cities) or economic development levels (χ2 = 342.20, 814.60 and 201.34, all P < 0.05). There was no significantdifference in the prevalence of human soil-borne nematode infections between male (18.21%, 441/2 422) and female residents (19.24%, 509/2 645) (χ2 = 0.89, P > 0.05), and soil-borne nematode infections were detected in residents at all age groups, with the greatest prevalence found in residents at ages of 1 to 9 years (25.88%). In addition, the highest prevalence of soil-borne nematode infections was seen in residents with the Dulong Ethnic Minority (82.09%), in preschool children (25.06%) and in illiterate residents (24.80%), and there was no age-, ethnicity-, occupation- or education level-specific prevalence of soil-borne nematode infections detected (χ2 = 46.50, 1 016.96, 36.33 and 52.43, all P < 0.05). CONCLUSIONS: The prevalence of human soil-borne nematode infections remains high in Yunnan Province. The management of soil-borne nematodiasis requires to be reinforced among low-age children, farmers, old people and residents with low educations levels or ethnic groups.

Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1.

DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells.

[Clinical significance of minimal residual disease in patients with Ph-negative precursor B-acute lymphoblastic leukemia].

Objective: To explore the predictive value of minimal residual disease (MRD) level in Ph-negative precursor B-acute lymphoblastic leukemia (ALL) patients. Methods: De novo 193 Ph-negative B-ALL patients from Sep 2010 to Nov 2017 were involved in the study. The patients' MRD evaluation which can be performed by multiparametric flow cytometry (MFC) after 1 month, 3-month, 6-month treatment. Relapse free survival (RFS) and overall survival (OS) were compared in patients with different MRD level. Results: The median follow-up was 22 months. All patients was evaluated at 497 MRD level. Patients who reach the good MRD level at 1 month (<0.1% or ≥0.1%), 3-month (negative or positive), 6-month (negative or positive) had a significantly higher probability of estimated RFS (74.5% vs 29.9%; 75.6% vs 29.7%; 74.6% vs 11.6%) and of estimated OS (67.5% vs 30.3%; 71.6% vs 27.8%; 74.0% vs 15.7%). Patients who reach the MRD negative at all 3 times had a significantly higher probability of estimated RFS (80.5% vs 30.5%) and better estimated OS (77.1% vs 29.4%) compared to patients with at least MRD failure in one time (P<0.001). Multivariable analysis showed MRD level at 3-month was an independent prognostic factor for DFS and OS. Conclusion: MRD is an important prognosis factor for Ph-negative B- ALL patients.

The modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase is a negative regulator of estrogen receptor-mediated transcription upon alkylation DNA damage.

Cell proliferation requires precise control to prevent mutations from replication of (unrepaired) damaged DNA in cells exposed spontaneously to mutagens. Here we show that the modified human DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O(6)-alkylguanine (6RG) lesions by MGMT in the cells exposed to alkylating carcinogens, functions in such control by preventing the estrogen receptor (ER) from transcription activation that mediates cell proliferation. This function is in contrast to the phosphotriester repair domain of bacterial ADA protein, which acts merely as a transcription activator for its own synthesis upon repair of phosphotriester lesions. First, MGMT, which is constitutively present at active transcription sites, coprecipitates with the transcription integrator CREB-binding protein CBP/p300 but not R-MGMT. Second, R-MGMT, which adopts an altered conformation, utilizes its exposed VLWKLLKVV peptide domain (codons 98 to 106) to bind ER. This binding blocks ER from association with the LXXLL motif of its coactivator, steroid receptor coactivator-1, and thus represses ER effectively from carrying out transcription that regulates cell growth. Thus, through a change in conformation upon repair of the 6RG lesion, MGMT switches from a DNA repair factor to a transcription regulator (R-MGMT), enabling the cell to sense as well as respond to mutagens. These results have implications in chemotherapy and provide insights into the mechanisms for linking transcription suppression with transcription-coupled DNA repair.

Implication of localization of human DNA repair enzyme O6-methylguanine-DNA methyltransferase at active transcription sites in transcription-repair coupling of the mutagenic O6-methylguanine lesion.

DNA lesions that halt RNA polymerase during transcription are preferentially repaired by the nucleotide excision repair pathway. This transcription-coupled repair is initiated by the arrested RNA polymerase at the DNA lesion. However, the mutagenic O6-methylguanine (6MG) lesion which is bypassed by RNA polymerase is also preferentially repaired at the transcriptionally active DNA. We report here a plausible explanation for this observation: the human 6MG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is present as speckles concentrated at active transcription sites (as revealed by polyclonal antibodies specific for its N and C termini). Upon treatment of cells with low dosages of N-methylnitrosourea, which produces 6MG lesions in the DNA, these speckles rapidly disappear, accompanied by the formation of active-site methylated MGMT (the repair product of 6MG by MGMT). The ability of MGMT to target itself to active transcription sites, thus providing an effective means of repairing 6MG lesions, possibly at transcriptionally active DNA, indicates its crucial role in human cancer and chemotherapy by alkylating agents.

A method for simultaneous identification of human active and active-site alkylated O6-methylguanine-DNA methyltransferase and its possible application for monitoring human exposure to alkylating carcinogens.

Cells resist the major mutagenic effects of alkylating agents by the action of O6-methylguanine-DNA methyltransferase (MGMT), which transfers the alkyl (R) group of O6-alkylguanine, produced in DNA by these chemicals, to a cysteine residue in its active site (formation of R-MGMT). We demonstrate that cellular R-MGMT (which represents a record or memory within the cells exposed to these chemicals) can be assayed by its sensitivity toward proteolysis by protease V8. The possible use of this assay for monitoring exposure to alkylating carcinogens was investigated by using cultured cells and a preliminary study with the use of human blood from normal subjects and patients undergoing chemotherapy. Cultured cell experiments show that R-MGMT is sufficiently stable for the monitoring purpose and its level bears a dose-response relationship to the concentrations of the alkylating agent used. Interestingly, experiments with blood from patients undergoing chemotherapy show a gradual formation of R-MGMT in 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and an induced MGMT deficiency in cyclophosphamide-treated patients. The use of this methodology, which allows for the possible quantification of active MGMT (cellular DNA repair capacity) and R-MGMT (recent exposure) simultaneously, in monitoring human exposure to alkylating carcinogens is discussed.

Intracellular localization of human DNA repair enzyme methylguanine-DNA methyltransferase by antibodies and its importance.

The human DNA repair enzyme, methylguanine-DNA methyltransferase (MGMT, M(r) 21,000), which protects cells against the mutagenic effect of alkylating carcinogens, was found to be localized in the cell nucleus (except the nucleolus) by immunofluorescence staining using polyclonal and monoclonal antibodies. The supporting experiments came from differential staining of the MGMT-deficient (mer-) and -proficient (mer+) cells, Western blotting analysis, and specific antibody depletion studies with the immobilized fusion protein, GSTMGMT-glutathione-Sepharose. Its localization in the nucleus agrees with its biological function and possibly explains the ineffective protection of mammalian cells (mer-) transfected with the Escherichia coli MGMT genes from bifunctional alkylating agents.

Factors influencing the repair of the mutagenic lesion O6-methylguanine in DNA by human O6-methylguanine-DNA methyltransferase.

Oligodeoxynucleotides of various chain lengths (p(Bp)nB, n < or = 9) and the eight possible dinucleotide phosphates (pm6GpB and pBpm6G), each containing a single O6-methylguanine residue (m6G), were used to study the repair kinetics of this lesion by the cloned DNA repair proteins; human 21 kDa O6-methylguanine-DNA methyltransferase (MGMT), human 43 kDa glutathione-S-transferase fused MGMT (GSTMGMT) and the Escherichia coli 39 kDa ada protein. The observed second-order repair rate constants are dependent upon both the chain length of the oligonucleotide substrates for all three proteins and in the case assuming O6-methylguanine is similar to B). The differences observed in the ratios of the rate constants for the substrates with five and four base residues; 125 for the E. coli 39 kDa ada protein, 640 for the human MGMT and 27,800 for the human fusion protein GSTMGMT, suggest that the pentanucleotide phosphate containing this lesion is the "optimal" substrate for the proteins. Surprisingly, the human GSTMGMT is shown to be more effective in the repair of longer substrates with the second-order repair rate constants for TATA-Cm6GTATA being 6.16 x 10(6) for GSTMGMT, 2.00 x 10(6) for MGMT and 0.27 x 10(6) M-1 s-1 for the E. coli 39 kDa ada protein. Thus, the presence of an additional protein domain at the N terminus of human MGMT can alter its selectivity towards certain substrates. Although a number of peptide domains are conserved between the E. coli 39 kDa ada protein and phosphates can also be used to explain the observed sequence specific repair of this lesion within certain DNA sequences.

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes.

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.

Structure-related properties of the mutagenic lesion 6-O-methylguanine in DNA.

Chemical probing of the structures of a few very similar 30 base-pair duplexes containing a 6-O-methylguanine (meG) residue at the 16th position reveals that the modified base simultaneously perturbs the helical structure in two ways; it preferentially unstacks the 3' neighbouring base residue (thymine in this study) on the same strand and it unstacks the pyrimidine to which it is base-paired. Depending on its neighbouring 5' base residue and the base-pairing pyrimidine, this perturbation can extend to a few base-pairs in both 3' and 5' directions from the abnormal base-pair. These perturbations can be detected by cleavage at the site for the restriction enzyme MaeII. The unstaking of the C in the meG.C and A.C base-pairs may explain the de novo methylation of these helices by the human DNA-(cytosine-5-)methyltransferase. Interestingly, the kinetics of repair of the 6-O-methylguanine-containing dinucleotides by the cloned human methylguanine methyltransferase appears to be largely determined by the strength of the stacking interaction between the 6-O-methylguanine and the 5' neighbouring base.

Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs.

We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.

Intracellular Localization and intercellular heterogeneity of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.

Analysis of O6-methylguanine-DNA methyltransferase in individual human cells by quantitative immunofluorescence microscopy.

A quantitative assay of immunofluorescence is described that can be performed on individual cells from standard pathologic specimens using fluorescence microscopy. The technique has been applied to measurement of O6-methylguanine-DNA methyltransferase, a DNA repair protein that is a molecular marker for resistance to chloroethylnitrosources used in cancer chemotherapy. The immunofluorescence assay makes use of monoclonal antibodies with specificity for human transferase, fluorescence microscopy with digital imaging, fluorescent bead internal standards, and computerized image analysis. This method is specific for the transferase, produces results correlated with activity measurements, and yields new data about tissue heterogeneity and subcellular localization previously unavailable with standard assay methods.

Cutaneous sarcoidosis. Report of two cases and literature review.

Sarcoidosis is a multi-system granulomatous disorder of unknown cause that presents more frequently in young adults with bilateral hilar adenopathy, pulmonary infiltrates, and skin or eye lesions. The multisystem clinical manifestations of this disease are a diagnostic challenge to all physicians. Although the clinical and pathological characteristics of sarcoidosis are well described, the decision to treat and the optimal therapy are less well defined. We report two patients of systemic sarcoidosis with cutaneous manifestations of the disease. Gallium whole body scintigraphy was performed and showed increased activity over the bilateral pulmonary hilar region (lambda sign), lacrimal and parotid glands (panda sign).

Ethical practice in the management of impaired neonates--a Chinese viewpoint.

In recent years, the issue of whether medical care should be given to impaired neonates has given rise to widely divergent views among practitioners of medicine, law and ethics. The general public also holds wide-ranging opinions on this subject. The divergence centres on whether medical care should be given to seriously impaired neonates, on whom should make decisions in these matters, and who should perform acts of euthanasia, in accordance with the current climate of opinion in China. This opinion has been focused both on ethical principles and on relevant precepts relevant to the "value of life." The author presents his opinions and explanations on the rights of impaired neonates, the rights and duties of parents and medical staff, and his views on the application of beneficence relative to this subject.

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.

Formation of N-nitrosamines and N-nitramines by photolysis.

N-Nitrosamines are generated by the photolysis of neutral aqueous solutions of nitrite salts and heterocyclic amines. With nitrate salts, photolysis produces a mixture of N-nitrosamines and N-nitramines. The reactions occur with low (5 mmol/l) reagent concentrations under very mild conditions and are faster than the concurrent decomposition of N-nitrosamines and N-nitramines by photolysis. Furthermore, both N-nitrosamines and N-nitramines effect transnitrosation reactions under mild neutral conditions when photolysed in the presence of other amines.

Interaction of oligonucleotides containing 6-O-methylguanine with human DNA (cytosine-5-)-methyltransferase [published erratumm appears in Biochemistry 1992 Aug 4;31(30):7008].

Thirty-base-pair synthetic oligonucleotide duplexes containing a single meG.C (meG = 6-O-methylguanine) or A.C base pair at the 16th position (i.e., 5'-CCCGTTTAAATATACXTATACCCGGGTACC-3', where X = A or meG) were used to study de novo methylation by the purified human DNA (cytosine-5)-methyltransferase isolated from CEM cells. Both duplexes containing meG.C and A.C base pairs show enhanced methyl group acceptor properties. Subsequent introduction of hemimethylated sites at the 15th position of the top strand (the C residue next to the abnormal base pair) and the 7th, 15th (which represents the C residue in the 6meG.C and A.C base pairs), and 27th positions of the bottom strand were used to study the maintenance methylation of the hemimethylated duplexes by the methylase. This revealed striking differences in the rate, amount, and sites of methylation, which are dependent on the position of the hemimethylated site in the duplex. The possible mechanism of action of the methylase is discussed. The data show that 6-O-methylguanine residues in DNA can have other genetic effects apart from their miscoding behavior and that meG.C and A.C base pairs exert different effects in terms of methylation.

Specificities of human, rat and E. coli O6-methylguanine-DNA methyltransferases towards the repair of O6-methyl and O6-ethylguanine in DNA.

The behaviour of highly purified bacterial expressed rat O6-methylguanine-DNA methyltransferase (MGMT) towards the repair of CGCm6GAGCTCGCG and CGCe6GAGCTCGCG (km6G/ke6G = 1.45, where k is the second order repair rate constant determined, m6G and e6G are O6-methyl and O6-ethylguanine) is similar to that of E. coli 39kD Ada protein (km6G/ke6G = 1.6). However, the human MGMT is very different (km6G/ke6G = 163). The preferential repair of O6-ethylguanine lesion by the rat MGMT appears not to be related to the lack of the initiator methionine in the expressed protein since similar results were obtained from N-terminal Glutathione-S-transferase (GST) fused protein (GSTMGMT) which retains the methionine. The possible relationship between these findings and the differences observed in the primary amino acid sequence of these proteins is discussed. In addition the preferential repair of O6-ethylguanine substrate by the 39kD Ada protein as compared to the catalytic C-terminus alone (different by 134 times) suggests that the N-terminus plays a crucial role in the repair of O6-ethylguanine. This is in contrast to the minor effects of the GST domain when fused to the N-terminus of mammalian MGMT.