We describe in detail a direct assay for the substrate-inactivated DNA-repair enzyme, O6-methylguanine-DNA methyltransferase (O6-MT), which measures the transfer of radiolabelled methyl groups from a prepared O6-methylguanine-DNA substrate to the protein fraction of an enzyme-containing cell/tissue extract. This assay, a modification of a previously suggested method for monitoring O6-ethylguanine-DNA repair [Renard, Verly, Mehta & Ludlum (1983) Eur. J. Biochem. 136, 461-467], is sensitive, highly reproducible, accurate and is, as described here and relative to previously published methods, well suited for use with a large number of test samples. We identified two problems with the O6-[Me-3H]methylguanine-DNA substrate used in the present work and in other reported assay: firstly, that of progressively higher assay backgrounds with increasing age of substrate, which was nullified by once-only purification of the double-stranded substrate by hydroxyapatite chromatography; secondly, a substrate of high specific radioactivity (30 Ci/mmol), made with freshly prepared tritiated methylnitrosourea, behaved as a substrate of 5 Ci/mmol when referenced against radiolabelled O6-methylguanine-DNA made with either [3H]- or [14C]-methylnitrosourea at the lower specific radioactivities of 1 Ci/mmol and 61 mCi/mmol respectively. This apparently stemmed from the known instability of high-specific-radioactivity [3H]methylnitrosourea and indicated that an expected increase in sensitivity of the assay does not necessarily result from increasing the specific radioactivity of substrates above approx. 1 Ci/mmol. Although O6-MT was stable to preincubation at 25 degrees C, marked losses of activity were observed at 37 degrees C, and more so at 45 degrees C. Enzyme lability at the higher temperatures was not, however, seen during preincubation in the presence of its substrate. O6-[Me-3H]methylguanine-DNA, which apparently protected O6-MT against thermal inactivation. As previously seen with other human cells and tissues, extracts of human spleen in the present study showed wide interindividual differences in O6-MT specific activity (18-fold), which spanned the range 50-900 fmol/mg of protein. Cultured human lymphoblastoid Jurkat cells contained approx. 57,000 enzyme molecules/cell. Substrate-inactivated [Me-3H]methylated O6-MT was analysed by SDS/PAGE and electroblotting. The different but similarly sized forms of this enzyme that we previously detected in human spleen [Major, Gardner, Carne & Lawley (1990) Nucleic Acids Res. 18, 1351-1359] were clearly resolved by fluorography of electroblots, but only at considerable expense of time. As expected, scintillation counting of the protein extracted from gel slices and linear-wire scanning of enzyme-associated radioactivity on electroblots were quicker methods for detecting the [Me-3H]methylated inactivated O6-MT.
Download full-text PDF |
Source |
---|---|
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1151195 | PMC |
http://dx.doi.org/10.1042/bj2770089 | DOI Listing |
Chaos
January 2025
School of Mathematics and Statistics, University College Dublin, Dublin 4 D04 V1W8, Ireland.
Synaptic plasticity plays a fundamental role in neuronal dynamics, governing how connections between neurons evolve in response to experience. In this study, we extend a network model of θ-neuron oscillators to include a realistic form of adaptive plasticity. In place of the less tractable spike-timing-dependent plasticity, we employ recently validated phase-difference-dependent plasticity rules, which adjust coupling strengths based on the relative phases of θ-neuron oscillators.
View Article and Find Full Text PDFIssues Ment Health Nurs
January 2025
Department of Psychiatry, Ryhov County Hospital, Jönköping, Sweden.
Patient-Initiated Brief Admission (PIBA) is perceived as a constructive intervention. It remains uncertain whether PIBA contributes to healthier behaviors among its users. To comprehend patients' motivation to engage in health-promoting behaviors, it is essential to understand how various nursing interventions influence the behavior-specific thoughts and feelings that lead to healthy behaviors.
View Article and Find Full Text PDFChondrosarcomas are the second most common primary bone sarcoma. Due to chondrosarcomas relative resistance to chemotherapy and radiation, surgical treatment has become the mainstay treatment option. The purpose of our study was to understand the proportion of patients in this population who undergo non-operative treatment options secondary to various reasons and analyze the difference in survival as well as patient and cancer specific characteristics between the two groups.
View Article and Find Full Text PDFBlood
January 2025
University of Chicago, Chicago, Illinois, United States.
Most diffuse large B-cell lymphoma (DLBCL) patients treated with immunotherapies such as bispecific antibodies (BsAb) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative, multi-omic approach was applied to multiple large independent datasets in order to characterize DLBCL immune environments, and to define their association with tumor cell-intrinsic genomic alterations and outcomes to CD19-directed CAR T-cell and CD20 x CD3 BsAb therapies. This approach effectively segregated DLBCLs into four immune quadrants (IQ) defined by cell-of-origin and immune-related gene set expression scores.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
February 2025
Department of Physiology and Membrane Biology, University of California Davis, Davis, CA 95616.
The L-type Ca channel (Ca1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca flux that drives Ca-induced-Ca-release, Ca1.
View Article and Find Full Text PDFEnter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!