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.

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Assessment of chromatin activity in mouse and human tissues

Pancreatic deoxyribonuclease preferentially digests active genes during all phases of the cell cycle including mitosis. Recently, a DNAse I-directed in ~ nick translation technique has been used to demonstrate differences in the DNAse I sensitivity of euchromatic and heterochromatic regions of mitotic chromosomes. This ill ~ technique has been used in this study to ask whether facultative heterochromatin of the inactive X chromosome can be distinguished from the active X chromosome in mouse and human tissues. In addition to this, in ~ nick translation has been used to distinguish constitutive heterochromatin in mouse and human mitotic chromosomes. Based on relative levels of DNAse I sensitivity, the inactive X chromosome could not be distinguished from the active X chromosome in either mouse or human tissues but regions of constitutive heterochromatin could be distinguished by their relative DNAse I insensitivity. The use of !D situ nick translation was also applied to tissue sections of 7.5 day mouse embryos to ask whether differing levels of DNAse I sensitivity could be detected between different tissue types. Differences in DNAse I sensitivities were detected in three tissues examined; embryonic ectoderm, an embryo-derived tissue, and two extraembryonic tissues, extraembryonic ectoderm and ectoplacental cone. Embryonic ectoderm and extraembryonic ectoderm nuclei possessed comparable levels of DNAse I sensitivity while ectoplacental cone was significantly less DNAse I sensitive. This suggests that tissue-specific mechanisms such as chromatin structure may be involved in the regulation of gene activity in certain tissue types. This may also shed some light on possible tissue specific mechanisms regulating X chromosome activity in the developing mouse embryo.

Protein subcellular targeting inTrichoderma aggressivum f. aggressivum

Trichoderma aggressivum f. aggressivum is a filamentous soil fungus. Green mold disease of commercial mushrooms caused by this species in North America has resulted in millions of dollars in lost revenue within the mushroom growing industry. Research on the molecular level of T aggressivum have jus t begun with the goal of understanding the functions of each gene and protein, and their expression control. Protein targeting has not been well studied in this species yet. Therefore, the intent of this study was to test the protein localization and production levels in T aggressivum with green fluorescent protein (GFP) with an intron and tagged with either nuclear localization signal (NLS) or an endoplasmic reticulum retention signal (KDEL). Two GFP constructs (with and without the intron) were used as controls in this study. All four constructs were successfully transferred into T aggressivum and all modified strains showed similar growth characteristics as the wild type non-transformed isolate. GFP expression was detected from all modified T aggressivum with confocal microscopy and the expression was similar in all four strains. The intron tested in this study had no or very minor effects as GFP expression was similar with or without it. The GFP signal increased over a 5 day period for all transformants, while the GFP to total protein ratio decreased over the same period for all transformants. The GFP-KDEL transformant showed similar protein expression level and localization as did the control transformant lacking the KDEL retention signal. The GFP-NLS transformant similarly failed to localize GFP into nucleus as fluorescence with this strain was virtually identical to the GFP transformant lacking the NLS. Thus, future research is required to find effective localization signals for T aggressivum.

Letter - American soldier's account of battles at Chippewa, Lundy's Lane and Fort Erie, 1814

Transcript (original grammar and spelling retained): My dear wife I take this time to inform you that I am well hoping that these few lines will Reach you and find you the same I shall in form you of all our Battles that we have had sence I left home we crossed in to Canada the 2 day of July and took fort Erie on the 3 day of July without loss of one man. We then marched down to Chipway eighteen miles below the Fort Erie we got there on the forth day and had our first battle on the 5 day our loss was not jistly known But the inemy loss was double to ours. The 6 day we started with the 2 Brigade to make a bridge a crost the crick two miles a bove the fort in Building the Bridge the inemy Brought up their Canon and playd upon us with their artiliery a bout two hours We drove them from the fort our loss was none the inemy loss was nineteen ciled dead on the ground we then marched to Queenston when we got thare our inemy had fledfrom the fort we then remained thair to Queenston ten days then we marched down to Fort George But that caurdly Chaney did not a rive with the fleet so we had to return back to Queenston thare was a bout six hundred militia formed on the heights of land thay fired up on us from their pickets and retreated to the mane body our flankers ciled and wounded and took about twenty before they got to the Maine body we then marched up the hill they gave us two firs but did not damage and then retreated from the field we stayed there one knight and then marched to Chipway and stayed there one night and the next day just as the sun set the first Brigade marched up in order to give them Battle a bout two miles from the Crick and began the Battle the 2 Brigade has to March up to the Niagara path and ingaged them we charged up on their artlery and took all their Canon Miller commanded the four companys that charged....the battles lasted three hours and forty minutes our loss was about 8 hundred cild and wounded our inemies loss was a bout fourteen hundred cild and wounded the next morning we Marched up in order to give them Battle a gin but thay was afraid to ingage us we then marched to Fort Erie and went to fortiffing and made a strong place the inemy folered us up and Began to cananade and held it fifty three days thay a tacked the fort the fifteenth of august thay atacked a bout one hiour be fore day Light we saw them and Blue up our maggerzean & two hundred of our inemy our loss wasa bout forty cild and wounded and our inemy loss was a bout one thousand on the 7 Day of September we atacked them and took their batteries and Broke all their canon and drove them from the field our loss was a Bout two hundred cild and wounded our inemy loss was a Bout 8 hundred cild and wounded...we crossed in to Canada with five thousand and came out with fifteen hundred we then Marched to Sackett’s harbor....am well and harty for the present....a bout comming home it uncarting for there is not any....given this winter as yet But I shall try to Come home if I Can But if I Cant I want you should take good car of the Phiddness[?] I have not Received any Money as yet But soon as I do receive some I send some home. I want you should write to me as soon as you receive this and and how Much Stock you wintor I Received your Letter with Great pleasure I feel uneasy a bout you I am a frade that you are sick or dead this is from your husband Chase Clough

Isoflavone exposure throughout suckling results in improved adult bone health in mice

Exposure to isoflavones (ISO), abundant in soy protein infant formula, for the first 5 days of life results in higher bone mineral density (BMD),greater trabecular connectivity and higher fracture load of lumbar vertebrae (LV) at adulthood. The effect of lengthening the duration of exposure to ISO on bone development has not been studied. This study determined if providing ISO for the first 21 days of life, which more closely mimics the duration that infants are fed soy protein formula, results in higher BMD, improved bone structure and greater strength in femurs and LV than a 5-day protocol. Female CD-1 mice were randomized to subcutaneous injections of ISO (7 Q1 mg kg/body weight/day) or corn oil from postnatal day 1 to 21. BMD, structure and strength were measured at the femur and LV at 4 months of age, representing young Q2 adulthood. At the LV, exposure to ISO resulted in higher (P,0.05) BMD, trabecular connectivity and fracture load compared with control (CON). Exposure to ISO also resulted in higher (P,0.05) whole femur BMD, higher (P,0.05) bone volume/total volume and Q3 lower (P,0.05) trabecular separation at the femur neck, as well as greater (P,0.05) fracture load at femur midpoint and femur neck compared with the CON group. Exposure to ISO throughout suckling has favorable effects on LV outcomes, and, unlike previous studies using 5-day exposure to ISO, femur outcomes are also improved. Duration of exposure should be considered when using the CD-1 mouse to model the effect of early life exposure of infants to ISO.

On the equation of state and atomic mean square displacement of crystals

We have presented a Green's function method for the calculation of the atomic mean square displacement (MSD) for an anharmonic Hamil toni an . This method effectively sums a whole class of anharmonic contributions to MSD in the perturbation expansion in the high temperature limit. Using this formalism we have calculated the MSD for a nearest neighbour fcc Lennard Jones solid. The results show an improvement over the lowest order perturbation theory results, the difference with Monte Carlo calculations at temperatures close to melting is reduced from 11% to 3%. We also calculated the MSD for the Alkali metals Nat K/ Cs where a sixth neighbour interaction potential derived from the pseudopotential theory was employed in the calculations. The MSD by this method increases by 2.5% to 3.5% over the respective perturbation theory results. The MSD was calculated for Aluminum where different pseudopotential functions and a phenomenological Morse potential were used. The results show that the pseudopotentials provide better agreement with experimental data than the Morse potential. An excellent agreement with experiment over the whole temperature range is achieved with the Harrison modified point-ion pseudopotential with Hubbard-Sham screening function. We have calculated the thermodynamic properties of solid Kr by minimizing the total energy consisting of static and vibrational components, employing different schemes: The quasiharmonic theory (QH), ).2 and).4 perturbation theory, all terms up to 0 ().4) of the improved self consistent phonon theory (ISC), the ring diagrams up to o ().4) (RING), the iteration scheme (ITER) derived from the Greens's function method and a scheme consisting of ITER plus the remaining contributions of 0 ().4) which are not included in ITER which we call E(FULL). We have calculated the lattice constant, the volume expansion, the isothermal and adiabatic bulk modulus, the specific heat at constant volume and at constant pressure, and the Gruneisen parameter from two different potential functions: Lennard-Jones and Aziz. The Aziz potential gives generally a better agreement with experimental data than the LJ potential for the QH, ).2, ).4 and E(FULL) schemes. When only a partial sum of the).4 diagrams is used in the calculations (e.g. RING and ISC) the LJ results are in better agreement with experiment. The iteration scheme brings a definitive improvement over the).2 PT for both potentials.

The link between extracurricular activities and academic achievement for youth in grades 5 and 7 /

This study examined the link between involvement in extracurricular activities and academic success for 504 youth in grades 5 and 7, using the first-year survey data from a longitudinal study conducted by Youth Lifestyle Choices-Community University Research Alliance (YLC-CURA). Specifically, the study investigated whether a linear or curvilinear relation existed between extracurricular activities and academic achievement for both in- and out-of-school activities. It was hypothesized that stress may be a possible mediator in the link between extracurricular activities and achievement Results indicated that students in grades 5 and 7 were involved in club and sport activities both inside and outside of school at fairly equal fi-equencies, with a mean frequency of approximately once a month. The hypothesis that a positive relation j between in- and out-of-school extracurricular activities and achievement was supported. The hypothesis that a curvilinear relation would exist between extracurricular activities and achievement was only supported for out-of-school activities. This finding supports the argument that too much or too little involvement in out-of-school activities is related negatively to a student's academic success; however, a moderate amount of involvement appears to be positive. The hypothesis that there would be a relation between involvement in extracurricular activities and stress level for both in-school and out-ofschool activities was not supported. Results were discussed in terms of educational implications and community resources for extracurricular activities.

Sensitivities of Ontario isolates of Venturia inaequalis to metiram /

The effects of metiram (Polyram 80 DF) on the growth of Venturia inaequalis, cause of apple scab, and the degradation of metiram were examined in culture media. Samples of V. inaequalis conidia were collected from nine orchards in 1998 and six orchards in 1999 and tested for sensitivity. Samples were plated on water agar amended with metiram or mancozeb. Mean EC50 values (effective concentration of fungicide required to inhibit germination of half the conidia) for each population were calculated. The mean EC50 values for metiram ranged from 0.26 - 1.20 ^ig metiram a.i./ml, with differences (Student Newman Keul's Test (SNK), a=0.05) between populations. EC50 values for mancozeb ranged from 0.06 - 0.58 which were also different (SNK, a=0.05). Five of these populations were examined for mycelial growth sensitivity to metiram by testing 30 monoconidial isolates from each population on metiram amended potato dextrose agar. Mean EC50 values for populations were calculated and ranged from 3.44-5.94 |ig metiram/ml, and showed differences (Friedman Test, a=0.05). As the EC50 values obtained are far less than the concentrations applied in the field, results indicate that Ontario populations of V. inaequalis are still sensitive to metiram and mancozeb. The stability of metiram in PDA at 22°C was studied over a 10-day period. The initial concentration of metiram decreased by approximately 50% within the first day, and continued to decline slowly, to approximately 20% of the initial concentration. The factors possibly affecting initial metiram degradation, including agar, heat, and the use of glass or polystyrene Petri dish composition were examined. The effects from the polystyrene in the Petri dish composition were negligible, however more studies must be done to examine metiram degradation during the first 24 hours of preparation.

Laura Dexter letter, commenting on the McLeod murder trial, October 5, 1841

Alexander McLeod was Deputy Sheriff of the Niagara District during the Rebellion of 1837-38 in Upper Canada. On December 24, 1837, he became aware of a scheme by the rebels to use the American steamboat Caroline to provide supplies to William Lyon Mackenzie and his followers on Navy Island in the Niagara River. McLeod notified the authorities in Upper Canada about the situation. A few days later, on December 29, Commodore Andrew Drew led a band of men, including McLeod, to the American side, where the Caroline was moored at Schlosser’s Wharf. A scuffle ensued, in which American Amos Durfee was killed. The Caroline was then released from its moorings, set on fire, and drifted downstream before sinking. Many Americans viewed the incident as a violation of their sovereignty. Tensions between the United States and England were already strained over a boundary dispute between Maine and New Brunswick, and the situation with the Caroline further escalated the tension. McLeod was subsequently arrested in November, 1840 in Lewiston, NY and indicted for arson and murder. The British foreign secretary, Lord Palmerston, insisted that McLeod be released and could not be held personally responsible for the incident, as he was acting on orders from authorities in Canada. Eventually, McLeod was acquitted.

Letter to F.B. Day [from S.D. Woodruff]

Letter to F.B. Day (3 pages, unsigned) [from S.D. Woodruff] in which he says he is enclosing the $36.00 owed to him, Nov. 5, 1878.