Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Ecological interactions between zooplankton and photosynthetic bacteria in Crawford Lake, Ontario

The present study was carried out to test the hypothesis that photosynthetic bacteria contribute a large portion of the food of filter feeding zooplankton populations in Crawford Lake, Ontario. The temporal and spatial variations of both groups of organisms are strongly dependent on one another. 14 By using C-Iabelled photosynthetic bacteria. the ingestion and clearance rates of Daphnia pulex, ~. rosea, and Keratella spp were estimated during summer and fall of 1982. These quantitative estimations of zooplankton ingestion and clearence rates on photosynthetic bacteria comprised an original addition to the literature. Photosynthetic bacteria comprised a substantial portion of the diet of all four dominant zooplankton species. The evidence for this is based on the ingestion and clearance rates of the dominant zooplankton species. Ingestion rates of D. pulex and D. rosea ranged 5 5 -1 -1 - -- 5 - -- 5 from 8.3X10 -1 to 14.6XlO -1 cells.ind. hr and 8.1X10 to 13.9X10 cells.ind. hr • Their clearance rates ranged from 0.400 to 1.000 -1 -1 -1 -1 ml.ind. hr. and 0.380 to 0.930 ml.ind. hr • The ingestion and clearance -1 -1 -1 -1 rates of Keratella spp were 600 cell.ind. hr and 0.40 ul.ind. hr respectively. Clearance rates were inversely proportional to the concentration of food cells and directly proportional to the body size of the animals. It is believed that despite the very short reg~neration times of photosynthetic bacteria (3-8 hours) their population densities were controlled in part by the feeding rates of the dominant zooplankton in Crawford Lake. By considering the regeneration times of photosynthetic bacteria and the population clearance rates of zooplankton, it was estimated that between 16 to 52% and 11 to 35% of the PHotosynthetic bacteria were' consumed· by Daphnia· pulex. and Q.. rosea per day. The temporal and spatial distribution of Daphnia pulex, !.. rosea, Keratella quadrata, K. coChlearis and photosynthetic bacteria in Crawford Lake were also investigated during the period of October, 1981 to December, 1982. The photosynthetic bacteria in the lake, constituted a major food source for only those zooplankton Which tolerate anaerobic conditions. Changes in temperature and food appeared to correlate with the seasonal changes in zooplankton density. All four dominant species of zooplankton were abundant at the lake's surface (O-4m) during winter and spring and moved downwards with the thermocline as summer stratification proceeded. Photosynthetic bacteria formed a 2 m thick layer at the chemocline. The position of this photosynthetic bacterial J-ayer changed seasonally. In the summer, the bacterial plate moved upwards and following fall mixing it moved downwards. A vertical shift of O.8m (14.5 to 15.3m) was recorded during the period of June to December. The upper limit of the photosynthetic bacteria in the water column was controlled by dissolved oxygen, and sulfide concentrations While their lower limit was controlled by light intensity. A maximum bacterio- 1 chlorophyll concentration of 81 mg Bchl.l was recorded on August 9, 1981. The seasonal distribution of photosynthetic bacteria was controlledinpart' by ·theg.-"z1ai'_.Q;~.zoopl. ank:tCm;-.Qther -ciactors associated with zooplankton grazing were oxygen and sulfide concentrations.

Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.