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Biomedical subjects

J M Boyle

Publications and source records attributed to J M Boyle.

At least 73 records · Page 4Linked to original sources
PubMed1986

Evidence for the excision repair of O6-n-butyldeoxyguanosine in human cells.

The persistence of O6-n-butyldeoxyguanosine (O6-nBudG) in DNA, the presence of O6-alkylguanine DNA alkyltransferase (AT) activity in cell extracts, and cell survival following exposure to N-n-butyl-N-nitrosourea (BNU), have been measured in normal and xeroderma pigmentosum cell strains, both transformed and untransformed. The rates of removal of O6-nBudG did not correlate with AT activity but did correlate with the ability of strains to excise bulky DNA lesions. BNU and N-methyl-N-nitrosourea dose-response curves for cell killing suggests that both AT and excision may be involved in the repair of cytotoxic lesions.

Alkylation↗
PubMed1985

O(6)-methyldeoxyguanosine in oesophageal DNA among individuals at high risk of oesophageal cancer.

A radioimmunoassay using monoclonal antibodies (MAb) against 0(6)-medGuo and 0(6)-etdGuo enabled the presence of these DNA adducts to be detected in human tissues. With this methodology and with 1 mg of hydrolysed DNA it is possible to detect approximately 25 fmol 0(6)-medGuo or 12.5 fmol 0(6)-etdGuo per mg DNA. The analysis comprised a total of 37 human tissue specimens derived from patients who underwent surgery for cancer of the oesophagus in Linxian County (People's Republic of China); for comparison 12 tissue samples obtained from hospitals in Europe were similarly analysed. Seventeen samples from Linxian County showed a level of 0(6)-medGuo ranging from 15 to 50 fmol/mg DNA, 10 showed higher levels up to 160 fmol/mg DNA, and the remaining 10 samples were below the limit of detection. All the tissue samples from Europe showed levels below 45 fmol 0(6)-medGuo/mg DNA, 7 being below the limit of detection. No 0(6)-etdGuo was detected in any of the samples. 0(6)-alkylguanine DNA alkyltransferase activity observed in tissue extracts from Linxian County ranged from 190 to 326 fmol (mean values) of 0(6)-medGuo removed per mg protein and lower values were obtained for the removal of 0(6)-etdGuo. The findings obtained using this approach appear promising in assessing the role of alkylating agents, e.g. nitrosamines, in the aetiology of human cancer.

China↗
PubMed1985

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein.

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation. Both mabs inhibited the binding of cytochalasin B to the glucose transport protein, reducing the affinity of this binding by greater than 2-fold. Each mab labelled the transporter polypeptide on Western blots both before and after treatment of the protein with endoglycosidase F, indicating that the epitopes recognised were located on the protein moiety of the glycoprotein. However, the mabs did not bind to the large fragments produced by tryptic or chymotryptic digestion of the native protein, although both mabs were shown to bind to sites on the cytoplasmic surface of the erythrocyte membrane.

Animals↗
PubMed1985

Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells.

Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [3H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. During the first 24 h after MNU AII cells produced a peak of activity that was not seen with LS cells. A second peak was seen in both cell lines between 24 and 48 h following MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [14C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Concentrations of 3AB in the toxic range of 3-10 mM inhibited poly (ADP-R) synthesis but no degradation of the polymer was observed. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, we have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities.

Alkylation↗
PubMed1984

Duodenogastric reflux in patients with gastric ulcer disease.

We studied reflux of duodenal contents into the stomach in patients with gastric ulcers, patients with duodenal ulcers, and normal subjects. Duodenogastric reflux was assessed in the fasting state and after cholecystokinin octapeptide administration (0.02 micrograms/kg intravenously). Slight reflux was observed in the fasting state in all three groups. However, after cholecystokinin octapeptide administration, reflux was significantly greater in gastric ulcer patients than in control patients for pancreatic phospholipase A2 (p less than 0.01) and lysophosphatidylcholine (p less than 0.001). Also in gastric ulcer patients, the gastric contents were significantly more alkaline (pH 5.26 +/- 0.58, p less than 0.001) during duodenogastric reflux than in normal subjects (pH 3.65 +/- 0.50) or duodenal ulcer patients (pH 2.67 +/- 0.63). Our results suggest that reflux of both pancreatic and biliary secretions might contribute to the gastric mucosal injury in gastric ulcer patients and we postulate that pancreatic phospholipase A2 might have a greater role in this process than has been previously acknowledged.

Adult↗
PubMed1983

Acute upper gastrointestinal hemorrhage in patients with chronic renal disease.

In order to reassess the role of duodenal ulcers as a cause of acute upper gastrointestinal hemorrhage in patients with chronic renal failure, 20 consecutive patients with moderate to severe chronic renal failure and a comparison group of patients without renal disease who were seen for acute upper gastrointestinal hemorrhage were reviewed. Gastric bleeding sites (gastric ulcer in 35 percent and gastritis in 20 percent) rather than duodenal ulcers were the most common sources of bleeding and were significantly associated with the use of ulcerogenic drugs. Patients with renal disease in whom acute upper gastrointestinal hemorrhage developed had significantly more morbidity and a trend toward higher mortality than the comparison group of patients without renal disease. It is concluded that gastric mucosal lesions, at least in part due to the use of ulcerogenic drugs, are the most common cause of significant acute upper gastrointestinal hemorrhage in patients with chronic renal failure.

Duodenal Ulcer↗
PubMed1983

Radioimmunoassay of O6-methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo.

Mouse monoclonal and rabbit polyclonal antibodies have been prepared against O6-methylguanosine complexed with bovine serum albumin. In radioimmunoassay 50% inhibition of binding of [3H]O6-methyldeoxyguanosine (O6-MedG) was obtained with 0.3 pmol or 0.16 pmol unlabelled O6-MedG using monoclonal or polyclonal antibodies having affinity constants of 3.0 X 10(9) and 5.9 X 10(9) I mol-1 respectively. Cross-reactivity with normal nucleosides and other adducts was determined. Sensitivity and reproducibility were improved by chromatographic separation using an Aminex A6 column eluted with 10 mM NH4HCO3 buffer which allowed quantitation of one molecule O6-MedG per 10(7) molecules dG in 2 mg calf thymine DNA. Values for O6-MedG from 14 samples of rat liver and kidney DNA analysed by the method described and by radiochromatography on Sephadex G10 were almost identical (correlation coefficient, 0.98). A modified procedure for the purification of 5-25 micrograms DNA from 1-5 X 10(6) fibroblasts applied to polycarbonate filters was used in providing further validation of the RIA system by measuring the persistence of O6-MedG in cell lines of known repair capacity.

Alkylation↗
PubMed1983

Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens.

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethane sulphonate (EMS) in Chinese hamster V79 cells and the mutagen sensitive derivative V79/79 was investigated by measurement of five parameters: production of strand breaks in template DNA, incorporation of [3H]TdR, semi-conservative and repair synthesis, molecular weights of pulse labelled DNA after mutagen exposure (nascent synthesis) and molecular weights of DNA pulse labelled and chased after mutagen exposure (elongation and ligation). Equal template strand breakage was evident in both cell lines immediately after MNU and EMS exposure and by 4-5 h after MNU the extent of fragmentation was greater in V79/79 cells. After u.v. irradiation template fragmentation was evident in V79/79 but not in V79 cells, even though V79/79 cells failed to excise cyclobutane dimers and repair synthesis was demonstrable in V79 cells but not in V79/79 cells after exposure to all three mutagens. The rate of incorporation of [3H]TdR during semi-conservative DNA synthesis was inhibited equally in a dose dependent manner after u.v. and MNU exposure; incorporation by V79/79 cells was inhibited to a greater extent than by V79 cells after EMS exposure. Nascent DNA synthesis was suppressed more in V79/79 cells than in V79 cells after u.v. but to similar extents in both cell lines after MNU and EMS treatment. Pulse chase experiments indicated a lower rate of elongation of nascent DNA in V79/79 cells after MNU and u.v. exposure but little difference was detectable after EMS.

Animals↗
PubMed1982

Reduced histidine-rich glycoprotein levels in plasma of patients with advanced liver cirrhosis. Possible implications for enhanced fibrinolysis.

Histidine-rich glycoprotein is a 3.8s alpha 2-glycoprotein of human plasma originally isolated in 1972 [1,2]. The biologic function of histidine-rich glycoprotein, however, is unknown. A recent report suggests that histidine-rich glycoprotein binds to the high-affinity lysine-binding sites of plasminogen and that histidine-rich glycoprotein may retard fibrinolysis by interfering with the binding of plasminogen to fibrin [3]. We have measured the plasma titers of histidine-rich glycoprotein in normal subjects and patients with advanced hepatic cirrhosis by single radial immunodiffusion with a monospecific antiserum. The levels in 22 patients were 7.0 +/- 2.5 mg/dl (mean +/- SD), whereas those in 20 control subjects were 11.8 +/- 2.7 (p less than 0.001). Upon two-dimensional crossed immunoelectrophoresis, the pattern of histidine-rich glycoprotein in liver cirrhosis was similar to that of normal histidine-rich glycoprotein. Since histidine-rich glycoprotein seems to function as an antifibrinolytic agent, the decreased titers in cirrhosis may be one factor contributing to the enhanced fibrinolysis commonly seen in this disorder.

Adult↗
PubMed1982

Potentiation of cell killing by inhibitors of poly(ADP-ribose) polymerase in four rodent cell lines exposed to N-methyl-N-nitrosourea or UV light.

The sensitivities (Do-values) of the cytotoxic effect of MNU on four rodent cell lines were: mouse L1210, 0.07 mM; rat Yoshida sarcoma, 0.52 mM; Chinese hamster V79A, 0.70 mM and the UV sensitive, X-ray sensitive V79/79, 0.35 mM. The abilities of maximum non-toxic doses of the poly-(ADP-ribose) polymerase inhibitors, 5-methyl nicotinamide (5MeN), 3-methoxybenzamide (3MBA) and caffeine to potentiate this cytotoxicity and that of UV light in V79A and V79/79 was measured. The degree of potentiation (ratio Do without inhibitor/Do with inhibitor) was both agent and cell line dependent. In general the lymphoid cell lines L1210 and YS showed greater potentiation, up to 4-fold, than did the fibroblast lines V79A and V79/79. The use of inhibitors in pairs suggested that 5MeN and 3MBA affect one process whereas caffeine affects additional processes. The data provide further support for a role for poly(ADP-ribose) in DNA repair, but indicate that metabolic factors may modify the effectiveness of individual inhibitors of poly(ADP-ribose) polymerase in different cell lines.

Animals↗
PubMed1982

Relative sensitivity of V79 and V79/79 cells to spontaneous and induced mutation to 6-thioguanine and ouabain resistance.

The relative responses of V79 and V79/79 cells to mutation to 6-thioguanine (6TGR) and ouabain resistance (OUAR) have been compared in unmutagenized cells and after exposure to ethyl methanesulphonate (EMS), N-methyl-N-nitrosourea (MNU) and ultraviolet light. In the V79/79 cell line, the spontaneous frequency of 6TGR colonies but not of OUAR colonies was enhanced compared to that in V79 cells. This appears to be the result of a reduced growth rate and plating efficiency of V79/79 cells and does not reflect a real difference in spontaneous mutability. V79/79 cells were more sensitive than V79 to the cytotoxic effects of all three mutagens, but induced mutation by UV, EMS and MNU to 6TGR was similar in the two cell lines. The lack of a differential response for induced mutation to 6TGR may reflect: (a) differences in selective stringency of thioguanine in the two cell lines, and (b) the greater susceptibility of V79/79 cells to induced chromosome damage. The relative mutability of the two cell lines to OUAR was dependent on the mutagen used. V79 cells were significantly more mutable than V79/79 cells after MNU exposure, but the two cell lines were similar in sensitivity to EMS-induced mutation. After UV-irradiation, however, V79/79 cells were morem utable than V79 cells. The differential response of the two cell lines to MNU suggests that O6-methylguanine is potentially mutagenic in V79 cells but is both potentially lethal and potentially mutagenic in the more sensitive V79/79 cells. The absence of a differential response to EMS-induced mutagenesis suggests that methylated and ethylated bases are repaired differently in Chinese hamster cells. The hypermutability of V79/79 cells by UV-irradiation indicates that thymine dimers are potentially lethal and potentially mutagenic in both cell lines.

Animals↗
PubMed1982

Sensitive radioimmunoassays for O6-n-butyldeoxyguanosine, O2-n-butylthymidine and O4-n-butylthymidine.

Radioimmunoassays have been developed using monoclonal antibodies from hybridomas raised against bovine serum albumin conjugates of O6-n-butylguanosine, O2-n-butylthymidine riboside and O4-n-butylthymidine riboside. The assays showed 50% inhibition of binding of specific (( 3H]butyl-deoxynucleosides by 0.044, 0.069 and 0.45 pmole of cold O6-n-butyldeoxyguanosine, O2-n-butylthymidine and O4-n-butylthymidine respectively, corresponding to affinity constants of 2.7 x 10(10), 1.1 x 10(10) and 8.8 x 10(8) respectively. In competitive radioimmunoassays a similar degree of inhibition required approximately 10(7)-fold higher concentrations of normal deoxynucleotides. From the relative inhibitions produced by a wide range of alkylated and normal nucleosides and bases we conclude that each antibody primarily recognises butylation, as compared to other alkylations, at the sites specified by the immunogen. The radioimmunoassays should be suitable for the detection of these potentially promutagenic lesions in digests of DNA exposed to low (biological) levels of butylation.

Animals↗
PubMed1981

Application of the Farr assay to the analysis of antibodies specific for UV irradiated DNA.

In order to determine the optimum conditions for reactivity in the ammonium sulphate precipitation (Farr) assay was have studied the DNA binding properties of two antibodies raised against ultraviolet single stranded DNA (UVssDNA) complexed with methylated bovine serum albumin. In general the buffer composition, pH, temperature, and ionic strength conditions described for binding to undamaged DNA were found to be appropriate for binding to UV-irradiated DNA. However, some differences in detail were noted which indicate the necessity for checking the physical conditions of binding of individual antibodies. Mouse monoclonal antibody and rabbit polyclonal antisera bound to UVssDNA very rapidly, even when DNA and ammonium sulphate were added simultaneously, whereas this procedure prevented binding of rabbit antisera to UV-irradiated double stranded DNA. Incubation at 45 degrees C for 30 min inhibited binding by mouse antibody, and incubation at 37 degrees C for 60 min caused reversible dissociation of the DNA-antibody complex. The optimised Farr assay was used to define the antigen specificities of the antibodies. The mouse antibody specifically bound to UVssDNA, but not to ssDNA, double stranded (ds) DNA, or UVdsDNA, whereas the rabbit antisera bound to UVssDNA, ssDNA or UVdsDNA, but not dsDNA. The extent of binding of the mouse antibody was dependent on the UV dose to the antigen, as well as the antigen concentration, indicating that the Farr assay can form the basis of a quantitative assay for photoproducts in DNA.

Ammonium Sulfate↗
PubMed1981

Pretreatment of Chinese hamster v79 cells with MNU increases survival without affecting DNA repair or mutagenicity.

Exposure of Chinese hamster V79 cells to a non-toxic dose of N-methyl-N-nitrosourea, followed at intervals by exposure to toxic challenging doses of the same agent, resulted in increased survival of colony forming ability when these cells were compared with matched control cells that only received the challenging dose. The extent of the increase was dependent on the time interval between exposures, and rose to a maximum of about two-fold 5 days after the initial dose, declining slowly to control values on subsequent days. Whilst pretreatment enhanced survival, it altered neither the frequency of mutation to 6-thioguanine resistance, nor the formation or loss of 3-methyladenine, 7-methylguanine and O6-methylguanine. Modification of the conditions by which the initial dose was administered led to a reduction or abolition of the survival response. It is suggested that enhanced survival may result from alteration in the ability to recover from cellular damage rather than by improved DNA repair.

Animals↗
PubMed1981

Effects of 5-methylnicotinamide on mouse L1210 cells exposed to N-methyl-N-nitrosourea: mutation induction, formation and removal of methylation products in DNA, and unscheduled DNA synthesis.

The lethality of N-methyl-N-nitrosourea (MNU) to mouse L1210 cells, as determined by colon forming ability, was potentiated 2.8 fold by the addition of 1 mM 5'-methylnicotinamide (5MeN). When 5MeN was present throughout the expression and selection of 6-thioguanine resistant mutants, the MNU-induced mutation frequency was reduced in duplicate experiments from 15.6 and 12.0 to 7.0 mutants per 10(4) survivors per mM MNU. At the same level of survival, cells treated with 5MeN had approximately 12 times fewer mutants than untreated cells. The rate of removal of the promutagenic lesion O6-methylguanine from DNA was enhanced approximately 2-fold, whereas that of 7-methylguanine was unaffected by the incubation of MNU treated cells with 5MeN. Since 5MeN is a potent inhibitor of poly(ADP-ribose) polymerase, this may imply that in normal cells it is specific ADP-ribosylation of the repair enzyme causing the removal of O6-methylguanine, rather than a more general modification of chromatin structure, that limits the rate of repair of the promutagenic lesion. 5MeN also stimulated unscheduled DNA synthesis in MNU treated cells, implying that an earlier observation that 5MeN prevented rejoining of strand breaks induced by repair of alkyl lesions, probably resulted from inhibition of ligation and not the failure of DNA polymerase to replace bases removed by repair nucleases.

Animals↗