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Biomedical subjects

R E Parchment

Publications and source records attributed to R E Parchment.

32 records · Page 2Linked to original sources
PubMed1992

A free-radical hypothesis for the instability and evolution of genotype and phenotype in vitro.

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., an in vitro evolution, which includes crisis, spontaneous transformation ('immortalization'), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by the in vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause of in vitro evolution. We propose an oxyradical hypothesis to explain each of the stages of in vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.

Aneuploidy↗
PubMed1991

Hydrogen peroxide as a mediator of programmed cell death in the blastocyst.

Previous work identified in blastocele fluid a soluble activity which killed embryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H2O2 [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H2O2 capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H2O2 toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H2O2 production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.

Animals↗
PubMed1990

Amine oxidases, programmed cell death, and tissue renewal.

Embryonal carcinoma cells, with embryonic (ECaE) or trophectodermal (ECaT) potential, have been used in a colony assay to determine regulatory mechanisms in the blastocyst. The mechanism that regulates ECaE and results in chimera formation is dependent upon a soluble factor in blastocoele fluid and contact with trophectoderm. Two mechanisms contribute to the regulation of ECaT: one involves a factor in blastocoele fluid and the other contact with either trophectoderm or inner cell mass which results in differentiation of the cells into trophectoderm, and the other involves the killing of at least 40% of the cells by blastocoele fluid alone. This cytotoxic activity probably causes the programmed cell death that occurs in the inner cell mass during blastulation as it loses the potential to differentiate into trophectoderm. A toxic activity similar to that of normal blastocysts has been obtained from embryoid bodies. This activity is caused by amine oxidase-dependent catabolism of polyamines, and it is postulated that programmed cell death in the embryo and chalone activity in the adult may have similar mechanisms.

Animals↗
PubMed1990

Serum amine oxidase activity contributes to crisis in mouse embryo cell lines.

This paper reports the results of experiments to test the hypothesis that crisis of spontaneous transformation is caused by the hydrogen peroxide and/or aldehydes generated from endogenous polyamines by serum amine oxidase [amine: oxygen oxidoreductase (deaminating), EC 1.4.3.6]. After 4-5 weeks of culture, crisis occurred in 16 of 29 cell lines derived from limb buds of embryos from SJL/J, C3H, and CD-1 mice. In contrast, after the same time in culture but in medium supplemented with aminoguanidine, which inhibits serum amine oxidase, crisis occurred in only 1 of 41 cell lines. Protection against crisis was maximal in cell lines of SJL/J embryos, in which the incidence of crisis fell from 7 of 9 in untreated controls of 0 to 12 in the presence of 2 mM aminoguanidine. 2-Mercaptoethanol at 150-300 microM, which protects cells from serum amine oxidase-dependent polyamine toxicity, also protected the cell lines against crisis. These protected cell lines retained proliferative potential, diploid DNA content, and the mixture of cell types found in the primary cultures. These results indicate that cytotoxic catabolites generated by serum amine oxidase caused at least a large portion, but perhaps not all, of the cellular damage that leads to crisis in mouse embryo cell lines.

Aldehydes↗
PubMed1990

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid.

There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No trophectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.

Animals↗
PubMed1990

Evidence linking programmed cell death in the blastocyst to polyamine oxidation.

Programmed cell death occurs in the inner cell mass during blastulation concomitant with the loss of its trophectodermal potential, and blastocele fluid kills malignant inner cell mass cells with trophectodermal potential (ECa 247) but spares those with embryonic potential (P19). A previous study had shown that blastocele-like fluid from embryoid bodies of the teratocarcinoma C44 contains a low-molecular-weight cytotoxin that exhibits the same target-cell selectivity as normal blastocele fluid. The current paper shows that the preferential killing of cells with trophectodermal potential is caused by hydrogen peroxide generated during the oxidation of polyamines in the cyst fluid by amine oxidases. The greater resistance of cells with embryonic potential to hydrogen peroxide is due to glutathione-dependent mechanisms. These data lead to the conclusion that an amine oxidase in the blastocyst oxidizes polyamines in blastocele fluid, generating hydrogen peroxide which causes programmed cell death of normal and malignant cells with trophectodermal potential.

Animals↗
PubMed1989

Polyamine oxidation, programmed cell death, and regulation of melanoma in the murine embryonic limb.

The murine embryonic limb at day 14 of gestation suppresses tumor formation by melanoma cells. Conditioned media of embryonic limbs have been found cytotoxic for B16 melanoma cells. The cytotoxicity is due to the catabolism of polyamines in the limb bud extracts by an amine oxidase in the serum supplement of the culture medium. However, a polyamine oxidase activity, similar to that in adult rat liver, is also detectable in homogenates of embryonic limbs. Thus, the embryonic limb contains the necessary components to produce polyamine-derived cytotoxic metabolites, which are present at the time programmed cell death occurs. This leads to the hypothesis that injected melanoma cells are killed incidentally by the mechanism that mediates programmed cell death.

Amine Oxidase (Copper-Containing)↗
PubMed1989

Mechanism of programmed cell death in the blastocyst.

The malignant growth potential of embryonal carcinoma cells may be controlled by environmental factors. For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of malignant cells but rather may lose all aspects of the malignant phenotype and become apparently normal embryonic cells. Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these injected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an embryonal carcinoma that makes midgestation chimeras, was regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass (ICM). In contrast, ECa 247, which makes trophectoderm, was regulated by exposure to blastocele fluid plus contact with trophectoderm or ICM. During the course of these experiments, dead embryonal carcinoma and ICM cells were observed, and blastocele fluid was then shown to kill ECa 247 and normal ICM cells of early blastocysts with trophectodermal potential. P19 cells and ICM cells with potential to make the embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition from early (when ICM has the potential to make trophectoderm) to late (when the ICM lacks the potential to make trophectoderm). It is postulated that this programmed cell death is designed to eliminate redundant ICM cells with trophectodermal potential, and its mechanism of action is mediated by epigenetic factors in blastocele fluid.

Animals↗
PubMed1986

The use of galactosyltransferase to probe nitrocellulose-immobilized glycoproteins for nonreducing terminal N-acetylglucosamine residues.

We report the use of UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyl-transferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6-3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-beta-N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.

Acetylglucosamine↗
PubMed1984

Glutathione S-transferase isozymes of mouse intestine: differential induction by 2(3)-tert-butyl-4-hydroxyanisole.

Electrofocusing of mouse small intestine mucosal cytosol yielded two prominent peaks of glutathione S-transferase activity, at pH 8.7 and pH 9.0. These intestinal transferases were indistinguishable from corresponding liver transferases in immunological reactivities and in several other properties. Dietary t-butyl-4-hydroxyanisole increased intestine cytosol protein immunologically identical to the liver transferase with pI of 8.7 and also elevated markedly and preferentially the intestinal transferase activity peak at pH 8.7. Thus, the predominant transferases of mouse intestine appear identical to two of the liver isozymes, and t-butyl-4-hydroxyanisole enhances intestinal transferase activity mainly by increasing the quantity of the isozyme(s) of pI 8.7.

Animals↗
PubMedDate not supplied

Programmed cell death (apoptosis) in murine blastocysts: extracellular free-radicals, polyamines, and other cytotoxic agents.

Programmed cell death can also be caused by extracellular, soluble factors, some of which are not physiologic. In vivo in the murine blastocyst, apoptosis eliminates redundant cells of unneeded phenotypes (pre-trophectoderm) from the inner cell mass. Although only a few cells die and many survive, all of the cells are exposed to the extracellular H2O2 in blastocele fluid. The specific control of life versus death is due to the developmental expression of glutathione-dependent protective mechanisms in the surviving cells. Based on recent results, a model is proposed in which apoptosis and necrosis reflect different temporal points along a single cell death program.

Animals↗