[New maternal insurance increases freedom of choice and quality of care].
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Choice Behavior↗
Biomedical subjects
B Rydberg
Publications and source records attributed to B Rydberg.
Inactive O6-methylguanine-DNA methyltransferase in human cells.
A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.
Bacteriophage lambda↗
Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.
We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.
Adenine↗
Partial purification of a human DNA glycosylase acting on the cyclic carcinogen adduct 1,N6-ethenodeoxyadenosine.
We previously reported that a variety of human cells and tissues contained a Mr35,000 DNA-binding protein which selectively recognized a single 1,N6-ethenoadenine in a defined 25-base double-stranded oligonucleotide (B. Rydberg et al., Proc. Natl. Acad. Sci. USA, 88: 6839-6842, 1991). We now demonstrate that incubation of the same duplex with 50-fold partially purified binding protein from human placenta results in release of the free 1,N6-ethenoadenine base, indicative of DNA glycosylase action. This enzyme activity appears unique in that it excises a cyclic adduct resulting from a known human carcinogen.
Adenine↗
Human cells contain protein specifically binding to a single 1,N6-ethenoadenine in a DNA fragment.
A human DNA binding protein has been characterized from cell-free extracts of liver, placenta, and cultured cells. This protein, apparent molecular mass approximately 35 kDa, to our knowledge, does not resemble other proteins reported to bind to carcinogen-modified DNA. The probe used for characterization was a 25-base oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine (epsilon A), a product of vinyl chloride metabolism. When annealed to form an epsilon A.T or epsilon A.C pair, a strong affinity to the protein was observed, with a binding constant of approximately 1 x 10(9) M-1. In contrast, very little binding was found with an epsilon A.A pair and none was found with an epsilon A.G pair. This suggests protein recognition of a specific structural alteration. Other defined probes with alkyl adducts did not bind. In addition, the human cell extracts and a rat liver extract were found to nick specifically at the 5' side of the epsilon A adduct, which could indicate a possible associated repair activity.
Adenosine↗
Localization of the human O6-methylguanine-DNA methyltransferase gene to chromosome 10q24.33-qter.
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Bacterial Proteins↗
cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase. cDNA expression in Escherichia coli and gene expression in human cells.
The O6-methylguanine-DNA methyltransferases are the most common form of cellular defense against the biological effects of O6-methylguanine in DNA. By screening a cDNA library with oligonucleotide probes derived from the active site amino acid sequence of the bovine methyltransferase, we have isolated a cDNA clone for the human enzyme. The cDNA contains a single open reading frame encoding a protein of Mr 21,700 which exhibits considerable homology to three bacterial methyltransferases. When provided with an Escherichia coli lac promoter, the encoded polypeptide can be expressed in E. coli to produce an active methyltransferase which is indistinguishable in size from the protein from human cells. The enzyme expressed in this way is functional in vivo and protects an E. coli methyltransferase deletion mutant against the mutational and cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. The methyltransferase gene spans at least 15 kilobases and is located on human chromosome 10. Alkylating agent-resistant Mex+ cells which express the methyltransferase protein contain a methyltransferase mRNA of about 1 kilobase. However, this mRNA is absent from three alkylation sensitive Mex- human cell lines indicating that the regulation of methyltransferase gene expression in these cell lines may be transcriptional.
Amino Acid Sequence↗
Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.
An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.
Amino Acid Sequence↗
Dose-dependent effects of verapamil and nifedipine on in vivo platelet function in normal volunteers.
The effects of 1 week of treatment with low and moderate doses of verapamil or nifedipine upon platelet function has been studied in 12 healthy volunteers. The ex vivo platelet aggregation threshold for ADP or adrenaline was not altered by verapamil or nifedipine. The plasma concentrations of beta-thromboglobulin and platelet factor 4 were significantly reduced by low but not by moderate doses of verapamil and nifedipine. Low doses of verapamil and nifedipine inhibit in vivo platelet activity in healthy volunteers.
Adenosine Diphosphate↗
Clinical results 1-10 years after highly selective vagotomy in 306 patients with prepyloric and duodenal ulcer disease.
Overall clinical results were assessed 1-10 years after routinely performed highly selective vagotomy (HSV) in 326 consecutive patients suffering duodenal and prepyloric ulcer disease, operated on by 37 surgeons of varying seniority at a district general hospital. Three hundred and six (94 per cent) patients were followed up. No mortality was recorded after 326 primary and 50 reoperative procedures. Postoperative complications were few and diminished during the study period. Persistent moderate to severe dumping was found in 2.3 per cent of the patients; slight and periodic, but in no case disabling, diarrhoea in 11 per cent and gastric retention in 2.1 per cent. The overall clinical ulcer recurrence rate was 13.8 per cent, with a tendency towards better results during the second 5 year period. Significant differences were found between individual surgeons and groups of surgeons. Recurrences tended to be located more proximally than the primary ulcer. Fifty reoperations were performed in forty patients for ulcer recurrence (twenty-five), persistent ulcer-like symptoms (twelve) and gastric retention (three). When all recurrences and reoperations were classified as failures the symptomatic assessment according to Visick showed 67 per cent of the patients in grades I-II and 26 per cent in grade IV but the corresponding figures from the patients' own classification including the results of reoperations were 79 per cent and 7 per cent. It is concluded that for the time being HSV is the method of choice for the elective surgical treatment of duodenal and prepyloric ulcer disease.
Adolescent↗
Clinical results of reoperation after failed highly selective vagotomy.
The results after reoperation after failed highly selective vagotomy during a 10 year period have been reviewed retrospectively. Forty of 306 patients (13 percent) underwent reoperation due to recurrent ulcer (25 patients), severe dyspepsia without proved recurrence (12 patients), and gastric stasis without recurrence (3 patients). In the first two groups, 16 patients had a second vagotomy and 17 underwent partial gastrectomy, 10 with gastroduodenostomy and 7 with gastrojejunostomy. The need for a second reoperation was disquietingly high after both revagotomy (5 of 16 patients) and partial gastrectomy with gastroduodenostomy (4 of 10 patients). These results contrasted with a successful outcome in all seven patients who underwent reoperation with partial gastrectomy and gastrojejunostomy. At the time of follow-up, 85 percent of the reoperated patients (34 of 40 patients) were in Visick grade 1 or 2 as determined by their own judgement.
Dyspepsia↗
DNA strand breaks induced by low-energy heavy ions.
The DNA unwinding method was used to estimate DNA breakage in Chinese hamster cells exposed to heavy ions with LET in the range of 750-5000 keV/micron. Comparison of the primary induced unwinding rate per dose unit for ions with various track diameters but similar LET showed a pronounced influence on the track diameter. Low-energy ions, producing thin tracks with diameters (penumbra) in the submicrometer region, were almost two orders of magnitude less efficient than more energetic ions producing tracks with diameters of several micrometers and about three orders of magnitude less efficient than X-rays. For the thin tracks, clustering of breaks was indicated by comparison of the DNA unwinding rates in two different alkaline solutions. The results indicate that the unwinding rate cannot be used as a good measurement for DNA breaks in this case. The residual unwinding remaining after 4 h of repair at 37 degrees C correlated well with the ability of the various ions to produce cell-killing.
Animals↗
Ulcer healing and relapse prevention by ranitidine in peptic ulcer disease.
Ranitidine, 300 mg daily, was given to 92 patients with duodenal ulcer (DU), 38 with prepyloric ulcer (PPU), and 21 with gastric corporeal ulcer (GCU). The healing rates at 4 weeks differed for the different types of ulcers (P less than 0.01), being 91% for DU, 68% for PPU, and 81% for GCU. After established ulcer healing, maintenance treatment with either ranitidine, 100 mg twice daily or 150 mg at night, or placebo was given for 1 year or until ulcer relapse in a total of 108 patients--71 with DU, 24 with PPU, and 13 with GCU. There were no significant differences in relapse rates between the two groups treated with active drug or between the three ulcer groups. However, the overall relapse rate in the active drug groups was 16%, against 72% in the placebo group (P less than 0.001).
Antacids↗
Repair of DNA double-strand breaks in colcemid-arrested mitotic Chinese hamster cells.
Chinese hamster V79 cells blocked in mitosis were irradiated with 60Co gamma-rays and incubated for repair in the presence of colcemid. DNA strand breaks were measured using neutral sucrose gradient centrifugation or the alkaline unwinding technique. It was found that mitotic cells repair DNA double-strand breaks (as well as single-strand breaks) efficiently, with a rate similar to exponentially growing asynchronous cells. It is argued that the dense packing of the chromatin in the mitotic chromosome makes a recombinational repair mechanism unlikely.
Animals↗
Detection of DNA strand breaks in single cells using flow cytometry.
A preliminary method is reported of alkaline unwinding of DNA within single cells and quantitation of the single-stranded and double-stranded DNA with the fluorescent probe acridine orange. A suspension of alkali-treated cells is obtained and analysed by flow cytometry. An increase in the amount of single-stranded DNA is taken as an indication of strand breaks. An advantage of this method is that a large number of cells can be individually analysed for DNA strand breaks. A measurement of DNA content is also obtained, making it possible to discriminate between cells in various parts of the cell cycle.
Animals↗
Recurrences 1 to 10 years after highly selective vagotomy in prepyloric and duodenal ulcer disease. Frequency, pattern, and predictors.
Three hundred and six (94%) of all consecutive patients with prepyloric or duodenal ulcer disease undergoing highly selective vagotomy at a District General Hospital were followed up after 1 to 10 completed years of observation. The 5- and 10-year cumulative recurrence rates were 11.6% and 16.8%, respectively. These figures were not related to age, sex, duration of ulcer disease, or preoperative peak acid output. Prepyloric ulcers had a significantly higher recurrence rate than duodenal ulcers during the first 5 years but this difference was eliminated at 10 years. The recurrence rates varied highly significantly between different surgeons. The 5-year recurrence rate in patients operated during the first 5-year period amounted to 13.4% and was steadily increasing. A corresponding figure for those operated during the second 5-year period was 5.3% and remained constant after 3 years of observation. This difference might reflect an improved surgical technique initiated by repeated evaluation of the clinical results.
Adolescent↗
Endoscopic papillotomy in patients with gallbladder in situ. Is subsequent cholecystectomy necessary?
Endoscopic papillotomy was performed for choledocholithiasis in 22 high-risk patients with gallbladder in situ. The procedure was well tolerated by the patients and was performed without serious complications. Pathologic laboratory findings normalized in most cases during the first week after papillotomy. The median hospital stay was four days. Laparotomy was subsequently performed in five cases, in one because of impaction of a 3 cm stone two days after the papillotomy, and in four because of biliary symptoms during a five-year follow-up period. On the basis of experience with this patient series and recent reports in the literature, primary endoscopic papillotomy is recommended as the sole therapeutic procedure for high-risk patients.
Aged↗
Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction.
Incubation of DNA with S-adenosyl-L-methionine (SAM) in neutral aqueous solution leads to base modification, with formation of small amounts of 7-methylguanine and 3-methyladenine. The products have been identified by high performance liquid chromatography of DNA hydrolysates and by the selective release of free 3-methyladenine from SAM-treated DNA by a specific DNA glycosylase. We conclude that SAM acts as a weak DNA-alkylating agent. Several control experiments including extensive purification of [3H-methyl]SAM preparations and elimination of the alkylating activity by pretreatment of SAM with a phage T3-induced SAM cleaving enzyme, have been performed to determine that the activity observed was due to SAM itself and not to a contaminating substance. We estimate that SAM, at an intracellular concentration of 4 X 10(-5) M, causes DNA alkylation at a level similar to that expected from continuous exposure of cells to 2 X 10(-8) M methyl methane-sulphonate. This ability of SAM to act as a methyl donor in a nonenzymatic reaction could result in a background of mutagenesis and carcinogenesis. The data provide an explanation for the apparently universal occurrence of multiple DNA repair enzymes specific for methylation damage.
Chromatography, High Pressure Liquid↗