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Biomedical subjects

K A O'Brien

Publications and source records attributed to K A O'Brien.

8 recordsLinked to original sources
PubMed2001

Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines.

A panel of human B-lineage lymphoma cell lines differing in cancer drug-resistance status and Bcl-2/Bax expression were used to study the contribution of mitochondrial-based perturbations and regulation in differential induction of apoptosis. Mitochondrial dysfunction was induced in cells by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP) and the respiratory chain inhibitor antimycin A. Cells were then assayed for early changes in MAP kinase signaling and subsequent induction of apoptosis. The cancer drug-resistant cell lines EW36 and CA46, overexpressing Bcl-2 and deficient in Bax, respectively, were both resistant to mitochondrial toxicant-induced cleavage of poly(ADP-ribose) polymerase (PARP) and morphologically detectable apoptotic cell death. In contrast, cancer drug-sensitive ST486 cell line, with low Bcl-2 expression, was sensitive to PARP cleavage and apoptosis engagement. Interestingly, mClCCP induced twofold more apoptosis than antimycin A in the ST486 cells. Exposure to the mitochondrial toxicants resulted in the early and preferential activation of the ERK and p38 MAP kinase pathways in only the drug-sensitive ST486 cell line, with mClCCP more potent than antimycin A. Specific inhibition of the p38 pathway augmented baseline and mClCCP-induced apoptosis. These results show that multi-drug-resistant and -sensitive B-lineage cells are also resistant and sensitive to compounds inducing mitochondrial dysfunction. The differential sensitivity to mitochondrial toxicant effects involved regulation by MAP kinases, since ERK and p38 were found to be preferentially activated only in the drug-sensitive B-lineage cells. Modulation of the p38 signaling pathway altered the sensitivity of cells to mitochondrial stress and may play a more general role in regulating the sensitivity of B-lineage cells to drugs and environmental toxicants.

Antimycin A↗
PubMed1999

Casein kinase 2 binds to and phosphorylates BRCA1.

The BRCA1 gene encodes a complex protein that appears to be involved in some aspects of DNA repair, transcription, or cell cycle regulation. The phosphorylation of BRCA1 is enhanced following episodes of DNA damage or during cell cycle progression, indicating that phosphorylation may be an important regulatory mechanism. Through a yeast two hybrid assay, we found that the beta-subunit of casein kinase 2 (CK2) associated with a carboxy-terminal region of BRCA1. This association was much weaker with the same fragment bearing a missense mutation (M1775R) that has been identified in breast tumors. The interaction was also evident in Sf9 cells. Subsequent studies showed that BRCA1 was phosphorylated in vitro by CK2. An analysis by site directed mutagenesis of BRCA1 showed that in vitro phosphorylation by CK2 required a serine at aa1572. These data implicate CK2 as a potential mediator of BRCA1 activity.

Alkaline Phosphatase↗
PubMed1998

Molecular determinants of L-type Ca2+ channel inactivation. Segment exchange analysis of the carboxyl-terminal cytoplasmic motif encoded by exons 40-42 of the human alpha1C subunit gene.

Recently we have described a splice variant of the L-type Ca2+ channel (alpha1C,86) in which 80 amino acids (1572-1651) of the conventional alpha1C,77 were substituted by another 81 amino acids due to alternative splicing of exons 40-42. Ba2+ current (IBa) through alpha1C,86 exhibited faster inactivation kinetics, was strongly voltage-dependent, and had no Ca2+-dependent inactivation. An oligonucleotide-directed segment substitution and expression of the mutated channels in Xenopus oocytes were used to study the molecular determinants for gating of the channel within the 80-amino acid domain. Replacement of segments 1572-1598 or 1595-1652 of the "slow" alpha1C,77 channel with the respective segments of the "fast" alpha1C,86 gave rise to rapidly inactivating alpha1C,86-like channel isoforms. We found that replacement of either motifs 1572IKTEG1576 or 1600LLDQV1604 of alpha1C,77 with the respective sequences of alpha1C,86 caused strong but partial acceleration of IBa inactivation. Replacement of both sequences produced an alpha1C, 86-like fast channel which had no Ca2+-dependent inactivation. These results support the hypothesis that motifs 1572-1576 and 1600-1604 of alpha1C,77 contribute cooperatively to inactivation kinetics of alpha1C and are critical for Ca2+-dependent inactivation of the channel.

Amino Acid Sequence↗
PubMed1990

Induction of mutations in TK6 human lymphoblastoid cells by ethyl methanesulphonate, benzo[a]pyrene and benzidine.

The experiments described were carried out as part of the UKEMS Third Collaborative Trial. The method used was essentially that described by Thilly et al., based on a cloning assay using microtitre test plates. The induction of mutations at both the thymidine kinase (tk) locus and the hypoxanthine guanine phosphoribosyl transferase (hprt) locus was determined following exposure to ethyl methanesulphonate (EMS), benzidine (BZD) and benzo[a]pyrene (B[a]P). EMS was tested in the absence of a metabolic activation system (S9 mix), BZD was tested in the presence and absence of S9 mix and B[a]P was tested only in the presence of S9 mix. Incubation with EMS or B[a]P caused the induction of mutations at both the tk and hprt loci. The effects of benzidine were not reproducible, although some individual data points at the tk locus were found to be significant.

Animals↗
PubMed1989

Tolerance to multiple doses of the pulmonary toxicant, naphthalene.

Intraperitoneal administration of single doses of the volatile aromatic hydrocarbon, naphthalene, resulted in dose-dependent bronchiolar epithelial cell necrosis in mice. Twenty-four hours after a dose of 50 mg/kg, swelling of Clara cells with some exfoliation of epithelial cells was evident in half of the treated animals. At doses of 100 mg/kg small numbers of necrotic and swollen cells with pyknotic nuclei were observed. At 200 mg/kg there were substantial numbers of bronchiolar epithelial cells sloughed into the airway lumen, apical projections were virtually absent, and there were large numbers of cells with pyknotic nuclei. In contrast, bronchiolar airways from mice treated with naphthalene daily for 7 days at doses of 50, 100, or 200 mg/kg/day differed only slightly from controls. Significant protection to bronchiolar epithelial cell necrosis produced by 300 mg/kg naphthalene was afforded by seven daily injections of 200 but not 50 or 100 mg/kg naphthalene. A gradual recovery in sensitivity to the 300 mg/kg challenge dose of naphthalene was observed as the time between the last 200 mg/kg naphthalene dose increased from 24 to 144 hr. Daily administration of 200 mg/kg but not 50 or 100 mg/kg naphthalene for 7 days resulted in a selective decrease in the rate of formation of 1R,2S-naphthalene oxide by mouse lung but not liver microsomal enzymes. This selective decrease in pulmonary microsomal formation of 1R,2S-oxide continued in animals killed 48, 96, and 144 hr after the last administration of 200 mg/kg. Alterations in the rate of formation of reactive, covalently bound naphthalene metabolites in lung microsomes were not observed, nor were there any differences in the levels of covalently bound reactive metabolites in vivo between tolerant and control animals. These studies are consistent with other work showing that the lung loses susceptibility to the acute injury arising from repeated exposure to pneumotoxicants. In contrast to other studies with naphthalene where alterations in the levels of covalently bound reactive metabolites in the lung closely paralleled the extent and severity of bronchiolar injury, these studies clearly separate necrosis from covalent binding. Although the correlation was not absolute, it appears that formation of 1R,2S-oxide by microsomal enzymes in vitro is a better overall marker of decreased sensitivity to naphthalene-induced bronchiolar necrosis than is reactive metabolite binding either in vivo or in vitro.

Animals↗
PubMed1985

Differences in naphthalene-induced toxicity in the mouse and rat.

Following the intraperitoneal administration of naphthalene (200 mg/kg) to mice, the lung, in comparison with other organs, was selectively damaged. Histological examination of the lungs showed that it was the non-ciliated, bronchiolar epithelial cells (Clara cells) which were damaged. At higher doses (400 mg/kg and 600 mg/kg, i.p.), there was also damage to the cells in the proximal tubules of the kidney. In contrast to the effect in mice, doses of naphthalene as high as 1600 mg/kg (i.p.) caused no detectable pulmonary or renal damage in the rat. This difference in toxicity between the mouse and rat was reflected by the ability of naphthalene to more severely deplete the non-protein sulphydryls in the mouse lung and kidney than in those organs in the rat. In order to investigate the species difference in toxicity, the metabolism of naphthalene by lung and liver microsomes of the mouse and rat was studied. In all cases, naphthalene was metabolised to a covalently bound product(s) and to two major methanol-soluble products, which co-chromatographed with 1-naphthol and 1,2-dihydro-1,2-dihydroxynaphthalene. However, both the covalent binding and metabolism were approximately 10-fold greater in microsomes prepared from mouse lung compared with those from the rat. This observation may in part explain the difference in toxicity of naphthalene to the mouse and rat lung. As 1-naphthol is a major metabolite of naphthalene and previous work had suggested that most of the microsomal catalysed binding of naphthalene was due to further oxidation of 1-naphthol, the role of 1-naphthol in mediating the naphthalene-induced toxicity was investigated. In neither the mouse nor the rat did 1-naphthol cause a depletion of non-protein sulphydryl levels or tissue damage in the liver, lung or kidney. Thus the toxicity of naphthalene does not appear to be mediated via 1-naphthol.

Animals↗