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Biomedical subjects

D L Mitchell

Publications and source records attributed to D L Mitchell.

At least 127 records · Page 7Linked to original sources
PubMed1985

Further characterisation of a polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity.

Synthetic polynucleotides irradiated with far (254 nm) or near (320 nm) UV-light were used to characterise 3 different radioimmunoassay systems. Antiserum raised against DNA irradiated with a high dose of far-UV-light was found to have at least 2 antibody populations. A competitive assay in which the labelled antigen was irradiated at 254 nm was found to be specific for Pyr(6-4)Pyo adducts, the antibody-binding sites being sensitive to a secondary photolytic dose of 320-nm light. When the labelled antigen was irradiated with 320-nm light the assay was specific for cyclobutane dimers. This assay had the same specificity as one consisting of labelled DNA irradiated with 254-nm light and an antiserum raised against DNA irradiated at 320 nm in the presence of acetophenone. These assay systems were used to demonstrate the dose-dependence of the induction and photolytic degradation of Pyr(6-4)Pyo adducts by a near-UV-light source.

Animals↗
PubMed1985

(6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers.

A polyclonal antiserum raised against UV-irradiated DNA can be used to assay cyclobutane pyrimidine dimers and Pyr(6-4)Pyo photoproducts specifically by changing the nature of the 32P-labelled antigen. Pyr(6-4)Pyo photoproducts were removed faster than cyclobutane dimers in UV-irradiated human, hamster and mouse cells. Xeroderma pigmentosum cells from complementation groups A, C and D were deficient in the repair of both lesions.

Animals↗
PubMed1985

Role of chromatin structure in the repair of DNA photoproducts in mammalian cells.

Chinese hamster ovary cells were irradiated with u.v. light and chromatin was isolated and fractionated according to magnesium-solubility in low ionic strength conditions. The minor, magnesium-soluble chromatin fraction was demonstrated to be enriched by over three-fold in transcriptionally active sequences and had proteins associated with it characteristic of transcriptionally active chromatin. Using a sensitive radioimmunoassay for u.v.-induced DNA photoproducts, we have shown that there are no differences in either induction or repair of u.v. DNA photoproducts in chromatin fractions from cells irradiated at low u.v. fluence.

Animals↗
PubMed1983

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts.

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G1 cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.

Animals↗
PubMed1983

The use of an immunological probe to measure the kinetics of DNA repair in normal and UV-sensitive mammalian cell lines.

Chinese hamster ovary cells and human fibroblasts were used to study UV-light-induced repair replication and removal of antibody-binding sites. Whereas repair replication still continued 8 h post irradiation, removal of antibody-binding sites was 80% complete within 2 h and reached a plateau by 4 h. This was found to be independent of the method of DNA isolation. UV-hypersensitive CHO cells exhibited reduced levels of repair synthesis that closely correlated with the extent of removal of antibody-binding sites. XP group A, C and D cells, each of which had less than 15% of the level of repair synthesis found in the control fibroblasts, removed less than 30% of the antibody-binding sites. Group E cells demonstrated intermediate levels of DNA-repair capacity in both assays.

Animals↗
PubMed1982

Loss of thymine dimers from mammalian cell DNA. The kinetics for antibody-binding sites are not the same as that for T4 endonuclease V sites.

Antiserum specific for thymine-containing dimers was used to assay DNA isolated from ultraviolet-irradiated cells following different repair periods. A 50% loss in antibody-binding sites was evident 1 h post-irradiation, and within 4 h 80% of the sites were removed. This result contrasts with data obtained with dimer-specific T4 endonuclease V and does not appear to be due to masking of the dimers by repair enzymes. T4 endonuclease V treatment of ultraviolet-irradiated DNA at 0 degree C resulted in conversion of the thymine dimers to apyrimidinic sites. This did not result in loss of antigenicity in either PM2 or CHO cell DNA. Likewise, treatment of ultraviolet-irradiated CHO cell DNA with T4 endonuclease at 37 degrees C did not change its antigenicity. These results suggest that aglycosylation of the dimers is not responsible for their inability to bind dimer-specific antibody 2-4 h post-irradiation. The possibility that T4 endonuclease V and the antiserum have different specificities for different dimers is discussed.

Animals↗
PubMed1982

A maternal and child health program to prepare and support parents in the postpartum.

The Maternal and Child Health Program Kit has been developed to prepare and support parents in the postpartum. The kit responds to four objectives: to help families to get to know their babies; develop feelings of competence; set realistic expectations for the postpartum; and to identify and use supports and resources. In developing the kit, two target groups have been identified--the primary group, the parents; the transmitter group, the educators. The kit includes question booklets at the bedside that encourage mothers to ask questions; resource cards for health professionals that facilitate consistency in information giving; information sheets to provide concise information on topics that cover detailed information or emotional areas; four slide-tape shows on breastfeeding and a coloring book for siblings. The Maternal and Child Health Program Kit is currently being evaluated.

Breast Feeding↗
PubMed1981

The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA.

Antiserum was prepared in rabbits against ultraviolet-irradiated DNA. Using a 125I-labeled protein A binding assay it was shown to be specific for ultraviolet-irradiated DNA, binding increasing as a function of logarithmic increase in dose. This antiserum was then used to develop a radioimmunoassay in which the competition between labeled UV-DNA and unlabeled sample DNA for antibody binding sites is monitored. Using this system the specificity of the assay could be changed depending on the nature of the labeled probe. The inability of poly(dA-dT) . poly-(dA-dT), as compared with poly(dA) . poly(dT), to act as a competitive inhibitor established that the primary lesion recognized by the antiserum is the thymine dimer. This antigenic response did, however, depend on the presence of at least one nucleotide adjacent to the dimer. The sensitivity of the assay was optimized by using 32P-labeled plasmid DNA as competitive probe and is capable of detecting photodamage in cellular DNA at doses as low as 2.5 J . m-2.

Animals↗
PubMed1979

The recovery of mammalian cells treated with methyl methanesulfonate, nitrogen mustard or UV light. I. The effect of alkylation products on DNA replication.

CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.

Animals↗
PubMed1979

The recovery of mammalian cells treated with methyl methanesolfonate, nitrogen mustard or UV light. II. The importance of DNA repair prior to the initiation of S phase.

CHO cells were synchronized 2 G1 phase and treated with UV light or HN2. These treatments resulted in a dose-dependent reduction in the rate of DNA replication and cell survival. Holding UV-irradiated cells in G1 phase (in HU medium) for an additional 10 h prior to their release into S phase did not assist recovery as measured by either of these criteria. The survival of cells treated with HN2 was also not enhanced by this recovery period. However, following 2 X 10(-5) M HN2 the rate of DNA replication increased from 30% to 70% of the control level when the period in HU medium was extended to 14 h. The induction of cross-links following HN2 treatment of asynchronous cells was shown to be dose dependent. Subsequent incubation in fresh medium resulted in complete recovery within 20 h at concentrations of HN2 up to 10(-5) M, and at 2 X 10(-5) M HN2, 75% of the cross-links were removed at 14 h post treatment.

Animals↗