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Biomedical subjects

T Carter

Publications and source records attributed to T Carter.

At least 37 records · Page 2Linked to original sources
PubMed1998

UV sensitivity and impaired nucleotide excision repair in DNA-dependent protein kinase mutant cells.

DNA-dependent protein kinase (DNA-PK), a member of the phosphatidyl-inositol (PI)3-kinase family, is involved in the repair of DNA double-strand breaks. Its regulatory subunit, Ku, binds to DNA and recruits the kinase catalytic subunit (DNA-PKcs). We show here a new role of DNA-PK in the modulation of the process of nucleotide excision repair (NER) in vivo since, as compared with their respective parental cell lines, DNA-PK mutants (scid , V-3 and xrs 6 cells) exhibit sensitivity to UV-C irradiation (2.0- to 2.5-fold) and cisplatin ( approximately 3- to 4-fold) associated with a decreased activity (40-55%) of unscheduled DNA synthesis after UV-C irradiation. Moreover, we observed that wortmannin sensitized parental cells in vivo when combined with either cisplatin or UV-C light, but had no effect on the DNA-PKcs deficient scid cells. Despite a lower repair synthesis activity (approximately 2-fold) measured in vitro with nuclear cell extracts from DNA-PK mutants, a direct involvement of DNA-PK in the NER reaction in vitro has not been observed. This study establishes a regulatory function of DNA-PK in the NER process in vivo but rules out a physical role of the complex in the repair machinery at the site of the DNA lesion.

Androstadienes↗
PubMed1998

Analysis of atypical squamous (glandular) cells of undetermined significance smears by neural network-directed review.

OBJECTIVE: The objective of this study was to evaluate the value of neural network-directed review of smears determined to contain atypical squamous (glandular) cells of undetermined significance to identify those cases most likely to be associated with cervical intraepithelial neoplasia. METHODS: One hundred sixty smears reported as atypical squamous (glandular) cells of undetermined significance on patients having colposcopy and directed biopsy within 1 year of the smear were identified. The smears were subjected to a neural network-directed review and classified according to findings on this review. The latter findings were related to those obtained on cervical biopsy. RESULTS: One hundred sixty smears originally reported as atypical squamous (glandular) cells of undetermined significance were subjected to neural network-directed review. The smears were upgraded in 20.6% of cases. Ninety-one patients were found to have normal biopsies, and 69 had biopsies reported as abnormal. Of the smears in patients with abnormal biopsies, 37.7% were upgraded, whereas only 7.7% of smears from those with normal biopsies were upgraded (P < .001). Nine patients were found to have cervical intraepithelial neoplasia-3 on biopsy. Six of the nine smears (66.7%) taken on these patients were upgraded. CONCLUSION: Neural network-directed analysis of smears conventionally diagnosed as atypical squamous (glandular) cells of undetermined significance will reveal findings suggesting a squamous intraepithelial lesion in a significant number of cases. This approach requires further study because it is a relatively cost-effective means of triaging patients with a cytologic diagnosis of atypical squamous (glandular) cells of undetermined significance.

Adolescent↗
PubMed1998

Transmyocardial laser revascularization.

Transmyocardial laser revascularization is a new treatment modality for patients with chronic angina that is refractory to traditional management. A laser is used to create full-thickness channels through areas of ischemic myocardium. Transmyocardial laser revascularization is performed in an effort to improve myocardial oxygenation, eliminate or reduce angina, and improve functional status. Transmyocardial laser revascularization currently is undergoing clinical trials. Early results have demonstrated significant reduction in anginal symptoms. Long-term efficacy of transmyocardial laser revascularization remains to be determined.

Angina Pectoris↗
PubMed1996

DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease.

We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PKcs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PKcs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PKcs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PKcs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PKcs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PKcs.

Alkylating Agents↗
PubMed1996

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication.

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.

DNA Replication↗
PubMed1996

Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity.

Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs). These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity. These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.

Amino Acid Sequence↗
PubMed1996

The placebo effect in acute headache management: ketorolac, meperidine, and saline in the emergency department.

In a prospective, double-blind, randomized study, ketorolac 60 mg, meperidine 50 mg plus promethazine 25 mg, and normal saline given by intramuscular injection were compared as treatment for acute headache crises. Thirty patients (6 men and 24 women) presenting to an urban emergency department with any type of benign headache were randomized into three groups and filled out the McGill Short-Form Pain Questionnaire with a Pain Rating Index and a Visual Analogue Pain scale. They received one of the study medications and repeated the testing after 1 hour. The objective was to test the efficacy of ketorolac in this population. Separate analyses of the McGill Short-Form (Total, Sensory, Affective, and Pain Rating Index scales) and the Visual Analogue Pain scale responses showed that the three treatments produced a significant reduction in pain (P < .0001), but that pain reduction did not differ among the treatments. This profound reduction observed after administration of a placebo prevented accurate evaluation of the effects of ketorolac. The placebo response must be considered in the design of future trials using intramuscular medications in the acute intervention of headache crises. In addition, the use of a standard analgesic is necessary to demonstrate both assay sensitivity and magnitude of response to placebo.

Acute Disease↗
PubMed1995

DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect.

Severe combined immunodeficient (SCID) mice are deficient in a recombination process utilized in both DNA double-strand break repair and in V(D)J recombination. The phenotype of these mice involves both cellular hypersensitivity to ionizing radiation and a lack of B and T cell immunity. The catalytic subunit of DNA-dependent protein kinase, p350, was identified as a strong candidate for the murine gene SCID. Both p350 and a gene complementing the SCID defect colocalize to human chromosome 8q11. Chromosomal fragments expressing p350 complement the SCID phenotype, and p350 protein levels are greatly reduced in cells derived from SCID mice compared to cells from wild-type mice.

Animals↗
PubMed1995

Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole-cell patch-clamp recording.

The measurement of cytosolic free Ca2+ ion concentration ([Ca2+]) with low affinity Ca2+ indicators has advantages for kinetic studies of cytosolic [Ca2+] transients when compared with more commonly used high affinity Ca2+ indicators. Their dynamic range and linearity are better suited to measurement of high localised transient concentration changes that exist near sites of influx or release, and the additional buffering introduced by the indicator is minimised. The fluorescent indicator furaptra (magfura-2) has low affinity for Ca2+ (approx. 50 microM) and can be used conveniently with single wavelength excitation at 420 nm with the procedure described by Konishi et al. [6]. The application of this protocol in whole-cell patch-clamp recording permits calibrated measurements of [Ca2+] during an experiment with minimal distortion of the time course and amplitude of [Ca2+] transients. A simple and inexpensive analogue circuit is described for direct computation of [Ca2+] from furaptra fluorescence with single wavelength excitation and emission during whole-cell recording. Data are shown which compare furaptra and fluo-3 estimates of the time course and amplitude of [Ca2+] changes in vascular endothelia, Purkinje neurones and hepatocytes.

Aniline Compounds↗