The specific objectives of my Master's project have been two-fold: to characterize the expression of prostaglandin endoperoxide synthase-1 (PGHS-1) protein and mRNA within the context of megakaryopoiesis, and to identify growth factors capable of inducing such expression. We induced MEG-01 cells to differentiate into platelet-like structures by treating them with TPA (12-0-tetradecanoylphorbol-13-acetate). We found that PGHS-1 protein levels were him in the platelet like population whereas PGHS-1 mRNA levels were greatest in the adherent population. We screened a number of recombinant hematopoietic factors for the ability to induce PGHS-1 protein expression in MEG-01 cells. We found that the combinations IL-3/IL-11/GM-CSF/SCF/TPO, IL-11/GM-CSF/SCF/TPO, IL-11/GM-CSF/TPO, IL-6/IL-11/GM-CSF/SCF, IL-6/IL-11, IL-3/IL-6, and IL 6/GM-CSF could induce PGHS-1 protein expression, but only when incubated with MEG-01 cultures that consisted solely of adherent cells. In conclusion, we have revealed that PGHS-1 protein and mRNA expression correlate strongly with megakaryocyte differentiation, and that certain cytokines can act in concert to stimulate adherent MEG-01 cells to increase their expression of PGHS-1 protein. The combination of IL-6 and -11 not only stimulates MEG-01 cells to increase their expression of PGHS-1 protein, but also PGHS-1 mRNA. (Abstract shortened by UMI.)

The role of apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) in hepatic triglyceride (TG)-rich very low density lipoprotein (VLDL) assembly has been investigated. Rat hepatoma McA-RH7777 cells were used to characterize structural determinants within apoB that are required for VLDL assembly/secretion, and to define mechanisms through which MTP facilitates VLDL assembly/secretion. Working with McA-RH7777 cells stably expressing recombinant human apoB100 (representing the full-length apoB) and various carboxyl-terminally truncated apoB proteins, we found that the apoB forms equal to or shorter than apoB29 (representing the amino-terminal 29% of the full-length apoB) were unable to assemble VLDL, whereas those equal to or longer than apoB34 could. Detailed biochemical analysis using apoB100- and apoB48-transfected cells revealed that assembly of VLDL, irrespective of the apoB length, was achieved through the same general pathway. Three notable features associated with the VLDL assembly process in McA-RH7777 cells were: (a) VLDL assembly was absolutely dependent upon exogenous oleate, (b) VLDL assembly was achieved post-translationally, and (c) the hallmark of VLDL assembly was the incorporation of bulk TG. Using chemical inhibitors of MTP, we found that MTP activity was required in the assembly/secretion of TG-rich VLDL, and that the requirement of MTP activity was determined by the amount of TG utilized for lipoprotein assembly. Moreover, the accumulation and attainment of TG within microsomal lumen was a function of MTP activity. Thus, MTP plays a role in facilitating the accumulation of TG within microsomes, an event that can be separated from TG incorporation into mature VLDL but represents an indispensable requisite for the post-translational assembly of TG-enriched VLDL.

The 175 kD aortic carboxypeptidase-like protein (ACLP), suggested to be involved in smooth vascular muscle cell differentiation, has been shown to be expressed in 3T3-L1 preadipocytes. In this study we demonstrate that ACLP protein expression is transiently but significantly down-regulated by day 2 of an 8-day 3T3-L1 differentiation. ACLP protein down-regulation correlates with increases in cell number, suggesting a potential link between ACLP and clonal expansion. The transient modulation of ACLP is shown to be partly due to transcriptional regulation. Analysis of the individual components of differentiation cocktail indicate that all components are necessary to induce maximal ACLP downregulation and, as such, this event is differentiation-dependent. Although ACLP overexpression had no apparent effect on adipogenesis, its pattern of expression, unique to post-mitotic proliferation, indicates a potential role for ACLP in adipose tissue development and warrants further investigation to elucidate its function during preadipocyte differentiation.

Human cytomegalovirus (HCMV) hyperimmune globulins or intravenous immunoglobulin (IVIG) have been widely used as a means of passive immunoprophylaxis. The efficacy of these blood products has not been clearly demonstrated in terms of the correlation between anti-HCMV titres assayed by enzyme-linked immunoassay (ELISA) and viral neutralizing activity measured in vitro by plaque reduction assay. Recent advances in the characterization of HCMV surface glycoproteins have led to the identification of the immunodominant regions of these molecules. The mouse monoclonal anti-HCMV antibody CMVB1 was utilized to study the properties of a neutralization epitope. Chemical cross-linking experiments with a photoactivable, heterobifunctional and cleavable reagent identified a viral antigen with an apparent molecular weight of approximately 70 kDa. Microtitre plate binding assays verified that the epitope was unique to HCMV and not homologous to herpesvirus-1 or -2 (HSV-1, -2) antigenic sites. The lectin concanavalin A was found to partially block CMVB1 from binding to the viral epitope, suggesting that carbohydrate moieties may be part of the antigen. Lectin specific sugars such as D-glucose, D-mannose and N-acetylglucosamine did not block CMVB1 binding to HCMV. Cell signalling events induced by HCMV particle binding to human foreskin fibroblast cells were also examined. Central molecules involved in three major signal transduction pathways were studied. Tyrosine phosphorylation was investigated with immunoprecipitation and immunoblotting techniques. Intracellular cyclic adenosine monophosphate (cAMP) concentrations were measured by immunoassay and protein kinase C (PKC) activation was established by a peptide radiolabelling assay. With the methodologies utilized, no evidence of HCMV-induced tyrosine phosphorylation or elevated cAMP concentrations was detected. PKC activity was found to increase to levels typically observed in activated cells. (Abstract shortened by UMI.)

Exposure of rainbow trout, Oncorhynchus mykiss, to ion-poor water or softwater elicits several morphological and physiological adaptations. This thesis examines these changes. The effects of a naturally-induced chloride cell proliferation on gill morphology was investigated (Chapter 2) by acclimating fish to softwater ((Na$\sp+\rbrack$ = 0.055 mmol l$\sp{-1};$ (Cl$\sp-\rbrack$ = 0.029 mmol l$\sp{-1};$ (Ca$\sp{2+}\rbrack$ = 0.059 mmol l$\sp{-1};$ (K$\sp+\rbrack$ = 0.007 mmol$\sp{-1}).$ Following 1, 2, and 4 weeks exposure to softwater, the results of scanning electron microscopy revealed a doubling of the chloride cell fractional area (CCFA: percentage of gill epithelium surface covered by chloride cells) due to both an increase in the number of chloride cells and the apical exposed surface area of individual chloride cells. During normoxia, ventilation frequency and opercular displacement were significantly higher in the softwater trout; opercular displacement was similar in both groups. PaCO$\sb2$ and plasma HCO$\sb3\sp-$ concentrations were significantly lower in the softwater fish and the blood acid-base status was characterized by a mixed respiratory alkalosis and metabolic acidosis such that blood pH was not statistically different in the 2 groups. During hypoxia the ventilation frequency and amplitude increased in the control trout, whereas only the ventilation amplitude increased in the softwater acclimated fish. The rate of PaO$\sb2$ reduction during hypoxia was significantly greater in the softwater fish and at the most severe level of hypoxia PaO$\sb2$ was significantly lower in the softwater fish. This thesis demonstrated that respiratory gas transfer is impaired by chloride cell proliferation during hypoxia and that hyperventilation is a compensatory physiological adjustment for softwater fish. (Abstract shortened by UMI.)

Apoptosis is a physiological form of cell death and is the cellular basis of ovarian follicular atresia and luteal regression. Apoptosis has distinct morphological and biochemical features including the degradation of DNA by the action of a Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease. This characteristic DNA degradation (DNA "ladders") is evident in atretic follicles and regressing luteal tissue of diverse species. Hormones, growth factors and cytokines that induce or suppress follicular atresia or luteal regression also induce or suppress the characteristic apoptotic DNA degradation. In order to elucidate the cellular and molecular mechanisms of ovarian apoptosis, the Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease responsible for DNA degradation must be identified. The objectives of the present work were to identify the endonuclease responsible for ovarian apoptotic DNA degradation and its possible regulation during follicular atresia and luteal regression. Immature female rats were sequentially treated with diethylstilbestrol (DES; DES group; preantral follicles), DES+eCG (eCG group; antral follicles) or DES+eCG+hCG (hCG group; luteal tissue). There was a developmental pattern of ovarian endonuclease activity such that nuclei from the eCG- and hCG-, but not the DES-group. degraded their DNA in an apoptotic fashion when exposed to Ca$\sp{2+}$ and Mg$\sp{2+}$ in vitro. Nuclear protein extracts contained three nuclease activities: a Mg$\sp{2+}$-dependent 27 kDa protein which was active on single-stranded DNA and did not display a developmental pattern of activity, and a doublet of 32/34 kDa which was Ca$\sp{2+}$/Mg$\sp{2+}$-dependent, active on double- or single-stranded DNA and present in the extracts of eCG and hCG, but not DES, groups. The 32/34 kDa activity was biochemically, immunologically and functionally indistinguishable from deoxyribonuclease I (DNase I). The mRNA encoding DNase I was also present, in a developmental pattern, in the rat ovary. Immunohistochemistry localized DNase I in nuclei of ovarian cells susceptible to apoptosis including luteal cells, granulosa cells from antral but not preantral follicles, and oocytes from preantral but not antral follicles. The possibility that caspase cleavage of actin and/or poly(ADP ribose) polymerase (PARP) regulates the activity of DNase I during ovarian apoptosis was investigated using an anti-eCG antibody in vivo to induce atresia and PGF$\sb{2\alpha}$ to induce luteal regression. Apoptosis during PGF$\sb{2\alpha}$-induced luteal regression, but not anti-eCG antibody-induced atresia, was associated with actin and PARP cleavage. Caspase-3 was immunolocalized in luteal cells and in theca, but not granulosa cells, of healthy follicles, and was also detected in granulosa cells of atretic follicles from anti-eCG antibody treated rats. These results demonstrate that the Ca$\sp{2+}$/Mg$\sp{2+}$-dependent endonuclease responsible for DNA "ladders" in ovarian cells is DNase I. Further, the expression and cellular distribution of DNase I during follicular development correlates with the susceptibility of ovarian cells to undergo apoptosis, which suggests that DNase I is responsible for the DNA "ladders" associated with follicular atresia and luteal regression. Although DNase I activity does not appear to be regulated by the caspase-mediated cleavage of actin or PARP, caspase-3 may be involved in the regulation of DNase I activity during ovarian apoptosis. Studies of the regulation of DNase I expression and activation will provide further insight into the cellular and molecular mechanisms of ovarian follicular atresia and luteal regression.

In developing neurons, microtubule-associated protein 2 (MAP2) occurs as high molecular weight MAP2 (HMW-MAP2) and low molecular weight MAP2 (MAP2c) isoforms. Though both HMW-MAP2 and MAP2c have been shown to stabilize microtubules (MTs) in vitro and in vivo, their developmental regulation has suggested that HMW-MAP2 may stabilize MTs to a greater extent than MAP2c. To test this hypothesis, MAP2c and HMW-MAP2 were independently transfected into undifferentiated P19 embryonal carcinoma (EC) cells using the constitutive phosphoglycerate kinase (PGK) promoter to drive expression. MT stability was assayed by treating transfected cells with the MT depolymerizing drug colchicine for various lengths of time and measuring the number of transfected cells possessing stable MT bundles. MAP2c and HMW-MAP2 stabilized MTs to similar extents suggesting that, in developing neurons, the increase in MT stability can not be attributed directly to the developmental exchange in MAP2c and HMW-MAP2 expression. Both MAP2c and HMW-MAP2 have been shown to promote the in vitro and in vivo assembly of MTs and inhibition of MAP2 expression in cultured neurons affects neuronal morphogenesis as indicated by a reduction in MT mass accumulation and neuritic growth. It is hypothesized that MAP2c and HMW-MAP2 are both required for neuronal morphogenesis. Comparison of relative MAP2 protein levels from untransfected and stable, MAP2cmyc-transfected neuronal cultures by Western blotting and ELISA showed higher MAP2c expression in the transfected cell line. MAP2cmyc expression was restricted to neurons and was first detected three days after neuronal induction at the time of neuronal outgrowth. In many neurons, MAP2cmyc was localized to all neurites and the morphology of these neurons appeared indistinguishable from untransfected, control neurons. (Abstract shortened by UMI.)

Reichert's membrane (RM) is a basement membrane that is deposited on the inner surface of the trophectoderm (TE) just prior to implantation in mice and rats. Components of RM include laminin, type IV collagen, fibronectin and heparan sulfate proteoglycan. The parietal endoderm (PE) cells have been shown to synthesize laminin, type IV collagen and heparan sulfate proteoglycan present in RM, while it appears that the fibronectin in RM is contributed by the TE cells. The PE, RM and TE together form the parietal yolk sac, which nourishes the developing fetus. The PE cells arise from the first inner cell mass (ICM)-derived cell lineage, the primitive endoderm (PrE) cells. The PrE cells move away from the ICM and migrate along the TE, differentiating to PE cells at some point in this migration. Primitive endoderm cells also secrete laminin and type IV collagen, and it is the contention of this author that secretion of these extracellular matrix (ECM) glycoproteins by the PrE cells may provide the substratum for their migration by contributing to the initial formation of RM. This research project studied the possible growth factor regulation of laminin glycoprotein secretion by PrE-like cells. (Abstract shortened by UMI.)

This thesis examines changes that occur in the epithelial cells of rainbow trout (Oncorhynchus mykiss) gills with respect to the expression of Na$\sp+$ and Cl$\sp-$ ion-transporting proteins during periods of acid-base disturbance. In the first part of the study, the expression of the V-type proton ATP-ase (H$\sp+$-ATPase) was examined in the gill of trout to provide direct evidence for its existence, determine its cellular distribution, and assess its response to acid-base disturbance. Gill epithelial cells demonstrated specific immunoreactivity, the intensity of which was increased markedly after 18 h of exposure to hypercapnia (1% CO$\sb2$ in air). The specificity of the antiserum for the 31-kDa subunit of the H$\sp+$-ATPase was supported by Western blotting with the presence of an immunoreactive band at 31-kDa. The increased H$\sp+$-ATPase immunoreactivity in the epithelial cells of hypercapnic fish demonstrates an "up-regulation" of this protein in response to respiratory acidosis. Oligonucleotide probes, complementary to the mRNA of the 31- and 56-kDa subunits of the bovine renal H$\sp+$-ATPase were constructed. The H$\sp+$-ATPase mRNA expression was increased markedly in the gills of acidotic fish and was accompanied by a pronounced elevation in branchial H$\sp+$ excretion. The probe for the Band 3 mRNA also demonstrated a specific hybridization signal in gill epithelial cells which was greatly increased in the gills of alkalotic fish, which were excreting large amounts of HCO$\sb3\sp-$. The results of this thesis provide direct evidence for the presence of an electrogenic H$\sp+$ pump located on the apical surface of the pavement cell which is actively up-regulated during periods of acidosis through increased transcription and translation of the protein as well as increased membrane association. Further evidence is provided for the presence of Band 3 or a Band 3-like protein which is responsible for Cl$\sp-$/HCO$\sb3\sp-$ exchange across the gill. (Abstract shortened by UMI.)

A cisplatin resistant cell line named E-8/0.7 was derived from the nonsmall cell lung cancer HTB 56 cell line. According to IC$\sb{50}$ estimations, E-8/0.7 cells were approximately 7.7 times more resistant to cisplatin than were HTB 56 cells. Uptake of cisplatin 100 $\mu$M over 1 hour was measured by atomic absorption spectrophotometry, and showed around a 30% lower uptake in E-8/0.7 cells compared to HTB 56 cells. Barotropic behaviour was analyzed by pressure tuning infrared spectroscopy. In the CH symmetric stretching region, we found a lower break point (and consequently lower membrane fluidity) in E-8/0.7 cells compared to the parent cell line. Lipid analyses of the two cell lines showed a higher cholesterol to phospholipid molar ratio in E-8/0.7 than in HTB 56 cells. Moreover, concentration of sphingomyelin was higher in E-8/0.7 cells than in HTB 56 cells.