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T A Bremner

Publications and source records attributed to T A Bremner.

9 recordsLinked to original sources
PubMed1997

A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro.

The apoptotic cysteine protease, caspase-3, is expressed in cells as an inactive 32-kDa precursor from which 17 kDa (p17) and 12 kDa (p12) subunits of the mature caspase-3 are proteolytically generated during apoptosis. Two amino acid sequences, ESMD downward arrowS (amino acids 25-29) and IETD downward arrowS (amino acids 172-176), in the precursor have been defined as the cleavage sites for the production of the p17 and p12 subunits. Using a cell-free assay system, we demonstrate that the caspase-3 precursor appears to be cleaved first at the IETD downward arrowS site, producing the p12 subunit and a 20-kDa (p20) peptide. Subsequently, the p20 is cleaved at the ESMD downward arrowS site, generating the mature p17 subunit. The cleavage at the IETD downward arrowS site required a protease activity that was selectively inhibited by the peptide, Ac-IETD-CHO (acetyl-IETD-aldehyde), and other protease inhibitors, such as the cowpox viral serine protease inhibitor, CrmA, and N-alpha-tosyl-L-phenylalanine chloromethyl ketone. The protease that catalyzed the cleavage at the ESMD/S site was selectively inhibited by another peptide, Ac-ESMD-CHO (acetyl-ESMD-aldehyde). More interestingly, the caspase-3 inhibitor, Ac-DEVD-CHO, but not the caspase-1 inhibitor, Ac-YVAD-CHO, also selectively inhibited the protease activity that cleaves at the ESMD downward arrowS site. This indicated that the cleavage at the ESMD downward arrowS site was either autocatalytic or that it required a caspase-3-like activity. In summary, we demonstrate that production of the p17:p12 form of caspase-3 is a sequential two-step process and appears to require two distinct enzymatic activities.

Caspase 3↗
PubMed1996

A decrease in glucose 6-phosphate dehydrogenase activity and mRNA is an early event in phorbol ester-induced differentiation of thp-1 promonocytic leukemia cells.

Redox modification of regulatory proteins implicates the glutathione redox system (GRS) in the control of gene expression. Glucose-6-phosphate dehydrogenase (G6PD) provides reducing equivalents for the GRS, and it has been suggested that high levels of G6PD in preneoplastic lesions are directly related to neoplastic transformation. We have used THP-1 human promonocytic leukemia cells, an established model of induced macrophage differentiation, to test an important corollary of this hypothesis, viz., that a decrease in G6PD activity should accompany the loss of the transformed phenotype. Phorbol 12-myristate 13-acetate (PMA) arrests the constitutive cycling of THP-1 and induces a phenotype that approaches normalcy. We measured the specific activities of the GRS enzymes, G6PD, glutathione peroxidase, and glutathione reductase during the early stages of phorbol ester-induced differentiation of THP-1 cells. We observed an 80% decrease in G6PD activity and an increase in the apparent KM for glucose 6-phosphate. In contrast, glutathione peroxidase (GPX) activity increased, while glutathione reductase (GR) activity remained essentially constant. The reduction in G6PD activity, preceding the loss of the transformed phenotype, is accompanied by a fourfold decrease in steady-state levels of G6PD mRNA. These findings are consistent with the hypothesis that high levels of G6PD are causally related to neoplastic transformations.

Blotting, Northern↗
PubMed1993

Phorbol myristate acetate-differentiated THP-1 cells display increased levels of MHC class I and class II mRNA and interferon-gamma-inducible tumoricidal activity.

The protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein induce differentiation of human monocytic leukemia (THP-1) cells along the monocyte/macrophage pathway of development. The differentiated cells express many important macrophage functions including phagocytosis and the secretion of immunomodulatory cytokines. Mezerein-differentiated THP-1 cells secrete interleukin-1 beta as well as a tumor cell growth inhibitory factor whose basal level is increased in response to interferon-gamma. However, tumoricidal, as opposed to tumoristatic, activity of differentiated THP-1 has not been documented. We report herein that PMA-differentiated THP-1 cells (PD/THP-1) contain elevated levels of MHC class I and class II mRNAs even in the absence of activating factors, and kill HT-29 human colon carcinoma cells when stimulated with recombinant human interferon-gamma. These two characteristics are important components of the macrophage phenotype. The results presented in this study extend previous observations on THP-1 cells by demonstrating that PD/THP-1 cells display a critical, immunologically relevant macrophage function, and therefore, enhance the utility of THP-1 as a model for the in vitro study of immunomodulatory drugs and macrophage-mediated cytocidal processes.

Antigens, Neoplasm↗
PubMed1985

Inverse effects of ethidium bromide on superoxide dismutase and lactate dehydrogenase of Artemia salina embryos.

Development of Artemia salina embryos in the presence of ethidium bromide, an inhibitor of mitochondrial transcription, results in a dose dependent increase in the specific activity of lactate dehydrogenase, and a concomitant decrease in the specific activity of a cyanide-resistant superoxide dismutase. The inhibition of mitochondrial function by ethidium bromide appears to exert opposite effects on the nuclear cistrons encoding lactate dehydrogenase and superoxide dismutase, and suggests that a common mitochondrial signal may exert diametric effects on nuclear cistrons whose products are characteristic of alternate states of respiration.

Animals↗
PubMed1985

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum.

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.

Catalase↗
PubMed1983

Estrogen regulation of superoxide dismutase in normal rat mammary tissues and mammary tumors.

Multiple electrophoretic molecular variants of superoxide dismutase were demonstrated in normal rat mammary tissues and DMBA-induced rat mammary tumors. The specific activities of Cu/Zu superoxide dismutase in mammary tumors of estrogen-treated rats were not significantly different from those activities seen in normal rat mammary tissues. However, the enzyme activities of mammary tumors from untreated rats (no estrogen) were significantly lower than the activities of normal rat mammary tissues. Exogenous estrogen appeared to raise superoxide dismutase levels in DMBA-induced rat mammary tumors to those levels seen in normal rat mammary tissues.

Animals↗
PubMed1978

Nucleoside phosphorylase 2 (Np-2) of mice.

Isozyme patterns of nucleoside phosphorylase (NP) in 16 inbred strains, two recombinant inbred, one congenic, and three species of wild mice were studied. Evidence is provided for a genetic locus, Np-2, encoding an electrophoretic variant which is expressed exclusively in erythrocytes of certain inbred strains. This finding establishes the occurrence of genetic polymorphism of NP among inbred strains of mice. In addition, the Npla allele previously reported only in inbred strains has been observed in one of the species of wild mice (Mus musculus castaneus) studied.

Animals↗