Dosimetry methods in photoradiation therapy.
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Antineoplastic Agents↗
Biomedical subjects
L R Wudl
Publications and source records attributed to L R Wudl.
In vitro studies of mouse embryos bearing mutations in the T complex: effects of culture in suboptimal medium upon t6/t6 and normal embryos.
We have compared the behaviour of normal and t6/t6 embryos in PCMF, a suboptimal 'delay' medium which arrests normal development. Morphologically, the two types of embryos are indistinguishable in this medium. Although beta-glucuronidase activity in embryos does not rise as quickly in delay medium as in cNCTC, a nutritive medium, the rate of increase is similar in t6/t6 and normal embryos. We conclude from these observations that the lethality of t6/t6 embryos is not a consequence of their reaching a given absolute age. Together with previous studies, our data suggest that embryo lethality correlates more closely with metabolic state than with morphological stage. Blastocysts maintained in PCMF are unable to give rise to trophoblast outgrowths but do so upon transfer into cNCTC medium. When a mixture of fourth-day t6/t6 and normal embryos are transferred to cNCTC medium after lengthy pre-incubation periods in PCMF, trophoblast outgrowth is initiated from both types of embryos at approximately the same time. However, when embryos are removed from the genital tract on the second or third day of gestation, t6/t6 embryos are slower to produce trophoblast outgrowths than are normal embryos upon transfer from PCMF to cNCTC medium. Although the reason for this differential behaviour is not yet clear, it is hypothesized that some product(s) required for the outgrowth process is (are) more unstable in third-day t6/t6 embryos than in normal third-day embryos or fourth-day t6/t6 embryos. Our ability to separate t6/t6 from normal embryos by their delayed initiation of trophoblast outgrowth provides us with a convenient way to identify, and to isolate for analysis, enriched populations of homozygous mutant embryos prior to the time at which they show gross morphological abnormalities.
Animals↗
H-2 analyses of blastocyst-derived cell lines from mice bearing T and tw18 mutations.
T/T and tw18/tw18 blastocysts appear to survive in culture for periods of 3 weeks at frequencies equal to that of normal embryos. We have, therefore, undertaken to generate cell lines with a homozygous mutant genotype. We attempted to identify these cells by H-2 haplotype analysis. The results indicate that barring recombination, which would be expected to occur at a frequency of about 15%, 1 of 10 blastocyst-derived cell lines from a (+/tw18 x +/tw18) cross had a tw18/tw18 genotype and 1 of 4 lines from a (T/+ x T/+) cross had a T/T genotype. Unexpectedly, all of the remaining lines were heterozygous; no +/+ lines were observed. The putative homozygous mutant lines might be useful in attempts to characterize tw18-specific antigens.
Absorption↗
In vitro studies of mouse embryos bearing mutations in the T complex: t6.
Cultured blastocysts homozygous for the t6 mutation lose their inner cell mass within a few days of attachment to the culture dish. At about the same time it becomes apparent that putative t6-mutant trophoblast cells and their nuclei fail to enlarge to the degree of their normal counterparts. These abnormalities in mutant embryos are reflected by an abrupt drop on the seventh equivalent gestation day in the rate of increase of beta-glucuronidase activity. The failure of t6/t6 trophoblast nuclei to enlarge normally appears to be due partially to endoreduplication at a slower rate than normal trophoblast nuclei and partially to premature cessation of DNA synthesis. Analyses indicate that this abnormality is not reversed when mutant embryos are placed in chimeric association with normal ones. Trophoblast outgrowths from mutant and normal trophectodermal vesicles are similarly distinguishable by differences in outgrowth and nuclear size as well as DNA content and synthesis. Despite the fact that t6/t6 embryos and trophectodermal vesicles are phenotypically different from normals from early times in culture, the trophoblast cells in the mutant structures acquire and continue to produce two enzymes characteristic of trophoblast differentiation, delta 5, 3 beta-hydroxysteroid dehydrogenase and plasminogen activator. On the basis of these and previous observations, we propose that the primary effect of the t6 mutation is to cause a metabolic lesion which kills inner cell mass cells relatively quickly but which has a more gradual effect upon trophoblast cells. The fact that phenotypically recognizable t6/t6 trophoblast cells can survive for several days before dying makes this a potentially useful system in which to search for the t6 gene product(s).
Animals↗
Nature of lethality of t mutations in embryos.
Mutations in the genes of the T complex in mice can lead to death at various stages of embryogenesis. It has been suggested that the T genes specify cell surface antigens which are essential for normal organisation of certain cells within the embryo. But, there is now evidence that at least some of these T complex mutations are simply generalised cell lethals.
Animals↗
In vitro studies of mouse embryos bearing mutations at the T locus: tw5 and t12 .
Lethal mutations in the T region of chromosome 17 in the mouse cause death at specific stages of embryonic development. It is widely believed that death of homozygous mutant embryos occurs as a result of aberrant cell-cell interaction followed by disorganization and death of specific cell types within the embryo. In vitro culture of blastocysts homozygous for one of these mutations, tw5, has revealed that this mutation is lethal for all cells of the mutant embryo. In addition, measurements of beta-glucuronidase activity indicate that the lethal period of the tw5 mutation is the same in vitro as it is in vivo. Similar biochemical studies with t12 mutant embryos demonstrate that cessation of macromolecular synthesis occurs rather abruptly at the morula stage, the time at which those mutant embryos die in vivo. These results lead us to propose that all cells within the conceptus are directly and profoundly affected by early acting t mutations, and that failure of some cell types to organize properly is not an adequate explanation of the death of these mutants.
Animals↗