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.

A comparative study of the change in diatom inferred ph of a staircase of lakes in the Algoma District, Northern Ontario /

Four staircase lakes occupying a single watershed located in the Algoma District, north of Lake Superior were chosen for this study. I examined the subfossil diatom assemblage in the top twenty centimeters of the surface sediments in each of these four lakes in an attempt to reconstruct their respective past pH history. From these analyses it was possible to test the hypothesis that the rate of change of diatom inferred pH was not significantly different in lakes located one below the other in a single "staircase" within a single watershed system. My results indicated that the four Z lakes had been acid for at least the last century. The water color of the three upper Z lakes (Z1, Z2 and Z3) was brown (>30 Pt Co units). The bottom lake (Z4) was the only clear water lake in the system «5 Pt Co units). This bottom staircase lake had no muskeg development around its shoreline. The alkaliphilous diatoms in the Z watershed system were important in determining the diatom inferred pH of the four Z lakes. The centric diatoms were extremely rare in the clearwater bottom lake (Z4). The ecology of the Eupodiscales is perhaps important in the interpretation of sediment in the more acid environment. Lake Z4 was the only one that had a progressive as well as a significant decrease in its downcore diatom inferred pH since the early 1960's. This lead me to speculate that the humic substances present in the upper three brown water lakes (Z1, Z2 and Z3) were perhaps Important in buffering them against a further decrease in water pH even though they were located within an area which was sensitive to acid precipitation.

The effects of yeast inoculation rate and acclimatization to juice on icewine fermentation /

Icewine is an intensely sweet, unique dessert wine fennented from the juice of grapes that have frozen naturally on the vine. The juice pressed from the frozen grapes is highly concentrated, ranging from a minimum of 35° Brix to approximately 42° Brix. Often Icewine fennentations are sluggish, taking months to reach the desired ethanol level, and sometimes become stuck. In 6 addition, Icewines have high levels of volatile acidity. At present, there is no routine method of yeast inoculation for fennenting Icewine. This project investigated two yeast inoculum levels, 0.2 gIL and 0.5 gIL. The fennentation kinetics of inoculating these yeast levels directly into the sterile Icewine juice or conditioning the cells to the high sugar levels using a step wise acclimatization procedure were also compared. The effect of adding GO-FERM, a yeast nutrient, was also assessed. In the sterile fennentations, yeast inoculated at 0.2 gIL stopped fennenting before the required ethanol level was achieved, producing only 7.8% (v/v) and 8.1 % (v/v) ethanol for the direct and conditioned inoculations, respectively. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 12.2% (v/v) ethanol, whereas the direct inoculum produced 10.5% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the rate of biomass accumulation, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was no significant difference in acetic acid concentration in the final wines across all treatments. Fennentations using unfiltered Icewine juice at the 0.5 gIL inoculum level were also compared to see if the effects of yeast acclimatization and micronutrient addition had the same impact on fennentation kinetics and yeast metabolite production as observed in the sterile-filtered juice fennentations. In addition, a full descriptive analysis of the finished wines was carried out to further assess the impact of yeast inoculation method on Icewine sensory quality. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 11.5% (v/v) ethanol, whereas the direct inoculum produced 10.0% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the peak viable cell numbers, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was a significant difference 7 in acetic acid concentration in the final wines across all treatments and all treatments affected the sensory profiles of the final wines. Wines produced by direct inoculation were described by grape and raisin aromas and butter flavour. The addition of GO-FERM to the direct inoculation treatment shifted the aroma/flavour profiles to more orange flavour and aroma, and a sweet taste profile. StepWise acclimatizing the cells resulted in wines described more by peach and terpene aroma. The addition of GO-FERM shifted the profile to pineapple and alcohol aromas as well as alcohol flavour. Overall, these results indicate that the addition of GO-FERM and yeast acclimatization shortened the length of fermentation and impacted the sensory profiles of the resultant wines.

A dimensional analysis of the benzylic hydroxylase active site in mortierella isabellina

The spatial limits of the active site in the benzylic hydroxylase enzyme of the fungus Mortierella isabellina were investigated. Several molecular probes were used in incubation experiments to determine the acceptability of each compound by this enzyme. The yields of benzylic alcohols provided information on the acceptability of the particular compound into the active site, and the enantiomeric excess values provided information on the "fit" of acceptable substrates. Measurements of the molecular models were made using Cambridge Scientific Computing Inc. CSC Chem 3D Plus modeling program. i The dimensional limits of the aromatic binding pocket of the benzylic hydroxylase were tested using suitably substituted ethyl benzenes. Both the depth (para substituted substrates) and width (ortho and meta substituted substrates) of this region were investigated, with results demonstrating absolute spatial limits in both directions in the plane of the aromatic ring of 7.3 Angstroms for the depth and 7.1 Angstroms for the width. A minimum requirement for the height of this region has also been established at 6.2 Angstroms. The region containing the active oxygen species was also investigated, using a series of alkylphenylmethanes and fused ring systems in indan, 1,2,3,4-tetrahydronaphthalene and benzocycloheptene substrates. A maximum distance of 6.9 Angstroms (including the 1.5 Angstroms from the phenyl substituent to the active center of the heme prosthetic group of the enzyme) has been established extending directly in ii front of the aromatic binding pocket. The other dimensions in this region of the benzylic hydroxylase active site will require further investigation to establish maximum allowable values. An explanation of the stereochemical distributions in the obtained products has also been put forth that correlates well with the experimental observations.

The role of personality and situational factors in forgiveness /

This study deals with personality and situational variables that influence forgiveness. The relations between empathy and forgiveness were studied, followed by the examination of the relation of these two variables to the Big Five personality traits, as well as honesty, absorption, the propensity to mystical experiences, and dissociation. Empathy was then tested as a mediating variable between the personality variables and forgiveness. Empathy and forgiveness were then studied in relation to childhood maltreatment. Finally, the effects of six different motivations to forgive were examined in relation to the personality variables. Participants were 142 undergraduate students recruited from the ftrst year psychology class at Brock University; 75% were either 18 or 19 years of age, and 84% were female. All of the variables were measured using self-report questionnaires. The relation between empathy and forgiveness was only partially replicated. In terms of personality, forgiveness was found to be related to honesty, emotionality, and agreeableness. Empathy at least partially mediated the relations between forgiveness and agreeableness, honesty and emotionality. Childhood maltreatment was negatively related to forgiveness, and positively related to openness to experience, absorption, and dissociation from reality, but not to the propensity for mystical experiences. Six different motivations for forgiveness emerged from an exploratory factor analysis. Out of these, Forgiveness to Promote Reconciliation was related to emotionality and dispositional empathy. Religious Forgiveness was related to honesty, emotionality, and mystical experiences. Forgiveness to Feel Better was related to honesty, emotionality, agreeableness, conscientiousness, absorption, mystical experiences, and empathy. Forgiveness to Assert Moral Superiority over the Injurer was negatively related to honesty, empathy, and positively related to extraversion. Forgiveness out of Fear was related to agreeableness. Finally, Altruistic Forgiveness was related to honesty, emotionality, and agreeableness, absorption and the propensity to mystical experiences. Altruistic Forgiveness correlated most highly with all the measures of forgiveness, followed by Forgiveness to Feel better. Altruistic forgiveness was also the motivation for forgiveness that correlated the highest with absorption.

DINOFLAGELLATE CYSTS FROM THE LATEST MIOCENE THROUGH MIDDLE PLEISTOCENE OF THE CARIBBEAN SEA, ODP SITE 1000: BIOSTRATIGRAPHY, PALEOCEANOGRAPHY, AND SHOALING OF THE CENTRAL AMERICAN SEAWAY

This is the first detailed study of organic-walled dinoflagellate cysts (dinocysts) and acritarchs for the latest Miocene–Middle Pleistocene of Ocean Drilling Program Site 1000 in the Caribbean Sea. Well-preserved and moderately diverse dinocysts and other palynomorphs reflect the interplay between neritic (carbonate-platform sourced) and oceanic species. The dinocyst biostratigraphy is tied to an existing marine isotope stratigraphy for the interval 5.5–2.2 Ma. For the interval 5.5–3.8 Ma, palynological samples are coupled to published sea-surface temperature estimates based on planktonic foraminiferal Mg/Ca. Changes in dinocyst assemblage composition are noted at ca. 4.6 Ma when shoaling of the Central American Seaway caused a temperature rise in the Caribbean, ca. 3.8–3.6 Ma, during the cold Marine Isotope Stage M2 when pronounced warming occurred, at ca. 2.7 Ma where possible weak cooling may reflect the onset of Northern Hemisphere glaciation, and in the Middle Pleistocene presumably reflecting global cooling and sea-level fall.