In this thesis, commitment is defined as the events that constrain a cell (and its progeny) to follow a particular developmental path, while differentiation is narrowly considered to involve events that result in attainment of a specific cell shape and does not consider functional or other characteristics. Therefore the P19 culture system was used to investigate the involvement of alpha and beta tubulin isoforms in formation of the stable microtubule (MT) array during neural differentiation. Two posttranslational modifications of alpha tubulin, acetylation and detryosination, are associated with stable MT populations including those of neural processes. Uncommitted embryonal carcinoma (EC) cells have minimal arrays of acetylated and detyrosinated MTs. Following neural induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment, before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine stable and only MT-associated protein (MAP) 1B is detected in association with this bundle. In cells which proceed to differentiate directly after commitment, this bundle apparently extends to form a neural process. MAP2C is first detected at about the time of neurite extension but is not limited to dendritic processes. During a brief period early in differentiation, indirect immunofluorescence staining shows the colocalization of colchicine stable acetylated MTs, vimentin and MAP2C. This thesis shows that acetylated MTs, MAP2 and vimentin intermediate filaments are arranged in an interdependent cytoskeletal array. This array may serve to stabilize processes in neural stem cells thereby allowing the stem cell to receive signals which influence the final decision to differentiate into neurons or glia. Finally, the involvement of beta tubulin isotypes in formation of the stable MT array during neural differentiation in P19 cells is analyzed by indirect immunofluorescence staining and by immunoblotting. (Abstract shortened by UMI.)

Our main objective was to determine if the Lymphokine Activated Killer cells (LAK) activity is restricted to Natural Killer (NK) cells or if it is also detectable in T cell subsets, especially in CD3+ T cells that are also negative for CD4 and CD8 cell surface markers. We were able to obtain highly purified subsets of NK cells, helper/inducer (CD3+4+8$-$), suppressor/cytotoxic (CD3+4$-$8+) and double negative (CD3+4$-$8$-$) T cells. The double negative T cells are now known to be a subset of $\gamma\delta$-T cells. Highly purified subsets were obtained using antibody (sheep anti-mouse IgG) coated magnetic particles and monoclonal antibodies. The purified subsets were tested for LAK activity against a standard target cell (K562), a NK resistant/LAK sensitive target cell (HTB 38), and a NK/LAK resistant target cell. We found NK cells develop LAK activity after IL-2 stimulation and that two T cell subsets (cytotoxic/suppressor and double negative) consistently showed LAK activity (3 experiments). The helper/inducer subset showed LAK activity in 2 of 3 experiments. Interleukin 4 had an inhibitory effect on the proliferative activity of IL-2 stimulated T cells and was not used in the LAK experiments. In conclusion we have found that the LAK function is a property of an heterogeneous group of lymphocyte subsets that includes NK cells and T cell subsets. (Abstract shortened by UMI.)

Mechano-sensitive ion channels in cells isolated from the heart and circumoesophageal ganglia of a freshwater snail (Lymnaea stagnalis) were characterized as K$\sp+$ selective and sensitive to changes in cell membrane tension. Using the technique of single-channel recording, stretch-activated K$\sp+$ (SAK) channels (30 pS in physiological saline) were shown to have complex permeation properties indicative of multi-ion channels. SAK channels were blocked by milli-molar quantities of quinidine or tetraethyl-ammonium and exhibited selectivity (Tl$\sp+ >$ K$\sp+ >$ Rb$\sp+ >$ Na$\sp+ >$ Li$\sp+$) properties seen in many other K$\sp+$ channels. SAK channels described in Lymnaea ventricular cells or neuron somas and growth cones were activated (increased open probability, P$\sb{\rm o}$) by increases in membrane tension produced by suction on the cell membrane. Stretch-activation was a reversible and repeatable phenomenon, associated with a reduction in a long closed time separating bursts of openings, rather than by an increase in the open time. The SAK channel's stretch-sensitivity was variable between patches, a finding attributable to the inability to accurately determine membrane tension. In spite of this, kinetic analysis of heart SAK channels established the presence of at least two open and three closed states. Similar analysis of neuron SAK channels indicated at least two open and four closed states. In channels from both cell types, only the long closed time constant ($\tau\sb{\rm C4}$, extracted from closed time frequency distributions) showed sufficient mechano-sensitivity to produce the observed increase in P$\sb{\rm o}$ with increasing membrane tension (in one patch, when P$\sb{\rm o}$ increased by a factor of 77 times the zero pressure P$\sb{\rm o}$, $\tau\sb{\rm C4}$ decreased by a factor of 22). In spite of the absence of specific SAK channel antagonists, attempts were made to establish a physiological role for SAK channels. Neurons and heart cells were shown to be resistant to severe hyposmotic insults. Hyposmotic shock resulted in hyperpolarization of the neuron soma resting potential and activation of SAK channels. These results suggested that SAK channels may contribute to osmotic-swelling limitation. SAK channels were found in developing neuron growth cones as well as the cell body. Channel characteristics were similar in both structures and circumstantial evidence points to expression of new SAK channels during neurite development. A new type of mechano-sensitive K$\sp+$ channel was found to coexist with SAK channels in neuron cell bodies and growth cones. These channels were in-activated (SIK) by increases in membrane tension and possessed a greater stretch-sensitivity than SAK channels. Decreased P$\sb{\rm o}$ was found to result from the lengthening of the long closed time in a fashion analogous but opposite to that seen in SAK channels, thus implying a common transduction mechanism. The differing stretch-sensitivities combined with similar permeation properties (6.6 pS in physiological saline; selectivity sequence, Tl$\sp+ >$ K$\sp+ >$ Rb$\sp+ >$ Na$\sp+$) suggested that membrane tension could control cell resting potential and thus modulate voltage-activated Ca$\sp{2+}$ channels and therefore the influx of Ca$\sp{2+}$, a known regulatory ion. SIK and SAK channels could therefore provide a mechanism for linking Ca$\sp{2+}$ influx to tension during axonal outgrowth.

The self-differentiative ability of the cranial neural crest cells in the Mexican axolotl, Ambystoma mexicanum was tested at various developmental stages. The processes through which cartilage determination occurs are far from complete; only a very small number of these cultures contained cartilage, although all appeared healthy and contained melanocytes and abundant mesenchyme. At all of these stages, pharyngeal endoderm is the only inductor required for cranial neural crest cells to differentiate into cartilage. Over 90% of cultures containing neural crest and pharyngeal endoderm produced mature cartilage nodules. The time course for chondrogenesis in vitro is similar to that which occurs in situ, which suggests that the culture system provides an accurate assessment of what occurs in vivo. Only those cells which normally contribute to skeletal elements have the capacity to do so. Neither trunk neural crest cells nor the cells of the transverse (anterior-most) neural fold can produce cartilage, even when placed in intimate contact with inductive tissues. Similarly, inductive endoderm is only found in the head, which is the origin of, and ultimate location of, all chondrogenic cells of neural crest origin. A sharp boundary, which occurs at the junction of the pharyngeal wall and the pharyngeal floor, divides the inductive from the non-inductive endoderm. All anterior endoderm is capable of inducing mature cartilage formation in virtually all cases, whereas cartilage formation occurs only rarely in the presence of posterior endoderm. Axolotl embryos which are homozygous for the premature death (p) gene do not develop past stage 37, which is well before cartilage differentiation usually occurs. They also display a variety of abnormalities in the gills, heart, pharynx, and liver, among others. Based on these and other observations, it was suspected that the mutation affected the endoderm. When mutant tissues were tested for the ability to form cartilage, however, the neural crest components of the culture, and not the pharyngeal endoderm, was found to be defective. The lack of cartilage is not caused by an abnormal localization of chondrogenic neural crest, nor does the migration of the cranial neural crest appear to be affected. The normality of the chordamesoderm and the ectoderm of mutant gastrulae was tested through the production of secondary neural structures. These studies determined that the chordamesoderm, which is the initiator of the events leading to neural crest specification, is normal in the mutant; the defect is in the actoderm of mutant embryos. The hypothesis that a number of neural crest functions are defective in the mutant appeared to be supported by the results of rescue attempts involving the transplantation of wild-type neural folds, and by the appearance of wild-type embryos which developed without neural crest cells. Thus, it would appear that the premature death mutation affects a subset of neural crest derivatives, and could therefore prove useful in the study of both the specification and differentiation of neural crest cells. (Abstract shortened by UMI.)

The adrenal medullary sigma and phencyclidine (PCP) receptors have been characterized by radioligand binding studies in bovine adrenal medulla. Using the prototypic sigma receptor ligand ($\sp3$H) (+)SKF-10047, two binding components were revealed, firstly, a high affinity sigma site and secondary, a low affinity "PCP-like" binding site. Investigation of the sigma and PCP-like binding sites in the adrenal medulla using the highly selective sigma ( ($\sp3$H) 3(+)PPP) and phencyclidine receptor ( ($\sp3$H) TCP) ligands has demonstrated that these two binding sites share many similar features. The radioligands ($\sp3$H) 3(+)PPP and ($\sp3$H) TCP saturate a similar maximal population of receptors (B$\sb{\rm max}$ = 218-250 fmol/mg protein) and both are most potently displaced by sigma ligands. Slight differences in stereoselectivity suggest that the binding of ($\sp3$H) TCP and ($\sp3$H) 3(+)PPP may be to multiple subtypes of sigma receptors. Evidence accumulated suggests that a novel peptide from adrenal chromaffin cell granules namely, histogranin, may act as an endogenous ligand for one or both sigma subtypes defined in this study. (Abstract shortened by UMI.)

Neuroblastoma is one of the most common paediatric solid tumours, frequently occurring in infancy with the primary lesion in the adrenal gland and sympathetic nervous system. It originates from primitive neural crest cells. In rare cases, this tumour regresses spontaneously to a more benign ganglioneuroma probably by neuronal differentiation or apoptosis. We investigated the role of induction of neuronal differentiation and apoptosis in vitro in SH-SY5Y neuroblastoma cells. A variety of agents that are known to induce neuronal differentiation including retinoic acid, nerve growth factor (NGF), protein kinase C (PKC) inhibitors and a cAMP-dependent protein kinase A (PKA) activator were tested solely or in combination for their capacity to induce terminal differentiation. The cells were characterised for markers of differentiation as well as their ability to withdraw from the cell cycle. We found that the combination of 8-Br-cAMP (PKA activator) with NGF in the presence of the cell cycle inhibitor aphidicolin was the best treatment to induce terminal differentiation in SH-SY5Y cells. Treated cells showed long neurites resembling those of neurones. They expressed markers characteristic of the cytoskeleton (NF200, NF68) and of neuronal function (tyrosine hydroxylase, choline acetyltransferase and the neurone-specific enolase) and showed an increase in the expression of TrkA, the receptor for NGF. Furthermore, the expression of the N-myc oncogene that is normally overexpressed in these cells decreased. Since neurones are dependent on NGF for survival, we tested the ability of terminally differentiated SH-SY5Y cells to survive in the absence of NGF. We found that the cells became NGF-survival dependent and that deprivation from this neurotrophic factor induced programmed cell death. We also tested the effect of PKC specific and non-specific inhibitors on cell proliferation, differentiation and apoptosis in SH-SY5Y cells. We found that only the non-specific PKC inhibitors staurosporine and H7 induced morphological and molecular differentiation as well as decreased N-myc amplification followed by apoptosis. The effect of differentiation induction on p53 sub-localisation was also investigated in undifferentiated and differentiated SH-SY5Y cells. p53 immunostaining showed that p53 was sequestered in the cytoplasm in undifferentiated SH-SY5Y cells and that the induction of differentiation resulted in the partial transfer of p53 to the nucleus where it probably became able to regulate the cell cycle and apoptosis since p53 is not mutated in neuroblastoma. Gap junction intercellular communication (GJIC) is known to be involved in the regulation of cell growth and homeostasis and has been shown to contribute to many diseases including cancer. We investigated the role of GJIC in neuroblastoma. We found that SH-SY5Y cells have altered GJIC due to an aberrant localisation in the perinuclear region of connexin 43 (Cx43), one of the proteins of GJIC normally present at the plasma membrane. GJ channel formation and gating is regulated by PKC, PKA and/or MAPK. We found that induction of differentiation using the PKA activator 8-Br-cAMP relocalised Cx43 to the plasma membrane region and restored GJIC. Furthermore, inhibition of p38 MAPK induced a block of cell proliferation associated with GJIC proficiency while inhibition of the subfamily of MAPK, Erk1/Erk2, likely promoted the degradation of Cx43.

Ku antigen (Ku70/Ku80) is a heteromeric, DNA binding protein that has been implicated in DNA repair, V(D)J recombination, tumour suppression, growth, cell cycle control, telomere maintenance and the regulation of gene transcription. Ku is a DNA binding subunit of the DNA-dependent protein kinase and functions to recruit and stimulate the catalytic subunit at appropriate DNA target sites. I present the unexpected finding that expression of human Ku fused to an ectopic activation domain broadly activated reporter genes integrated into the yeast genome independent of the reporter gene, yeast minimal promoter and reporter gene integration site. Interestingly, expression of various Ku70 deletion mutants alone or in combination with Ku80 revealed that the regions of Ku identified to be required for activation share a striking resemblance to portions of Ku needed to bind DNA. Furthermore, a mutation in Ku70 that negates the Ku70-dependent activation property in yeast also disrupts the ability of Ku70 monomers to associate tightly with the nucleus in mammalian cells. Moreover, I demonstrate that human Ku80 cannot substitute for the yeast homologue (HDF2) to rescue the temperature sensitive phenotype of a yeast strain deficient in Ku80 but expression of a variant human Ku heterodimer partially rescues the phenotype. By contrast, the characteristic slow growth displayed by HDF2 deficient yeast at 30°C could not be rescued by expression of the same human Ku heterodimer. Also, I determined that Ku70 contains a bipartite nuclear localization signal between amino acids 539--556, while the core of the nuclear localization signal of Ku80 is a basic motif from 565--569. Each nuclear localization signal mediated the nuclear transfer of the individual Ku subunits and Ku heterodimer, while site directed mutagenesis of both nuclear localization signals resulted in a Ku heterodimer that was localized to the cytoplasm. Lastly, I show that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization reduce the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

The kidney is believed to play a major role in the clearance and re-absorption of high density lipoprotein (HDL) particles from the blood. Experiments were undertaken to explore the specific sites of renal HDL metabolism in vivo and to investigate in vitro the factors that regulate the renal re-absorption of HDL by HKC-8 human proximal tubule (PT) cells. Perfusion of a rabbit renal artery with [3H]cholesteryl ester (CE) and 125I-protein labeled HDL particles showed that the kidneys are capable of filtering both apolipoprotein A-I (apoA-I) and whole HDL. A fluorescent microscopic study with the HKC-8 cells showed that the PT cells can bind and take up HDL particles. (Abstract shortened by UMI.)

Enterovirus 70 (EV70) belongs to the family Picomaviridae and is a human pathogen responsible for acute hemorrhagic conjunctivitis. Unlike most human enteroviruses, the host range of EV70 encompasses a wide variety of non-primate mammalian cells in vitro. We demonstrated previously that EV70 uses decay-accelerating factor (DAF/CD55), a 70 kDa glycosyl-phospatidylinositol (GPI)-anchored protein, as a receptor on HeLa cells. Others have shown that DAF also facilitates the attachment of other human enteroviruses, including a number of coxsackieviruses and echoviruses; however, for some of these viruses additional surface proteins are essential for virus entry and infection, such as intercellular adhesion molecule 1 (ICAM-1) for coxsackievirus A21, and the coxsackie-adenovirus receptor (CAR) for coxsackie B viruses. One objective of the research described in this thesis was to determine if human DAF also functioned as a receptor for EV70 on other human cell lines. Another objective was to determine whether ICAM-1 or human CAR function as receptors for EV70. A third objective was to elucidate the potential role of non-primate DAF homologues in EV70 binding and infection. (Abstract shortened by UMI.)

Recent studies demonstrated a role for annexin II (A2) and its ligand p11 in fibrinolysis. Since thrombin is a potent cell modulator obligately produced at the site of clot formation, the hypothesis was addressed that thrombin regulates fibrinolysis by increasing cell surface A2 and p11. Immunofluorescence and biotinylation established that thrombin significantly increases both on human umbilical vein endothelial cell (HUVEC) and human foreskin fibroblast surfaces. Intracellular antigenic analyses revealed increased p11 but unchanged A2, suggesting that transbilayer trafficking of A2 is controlled by p11. The thrombin receptor activating peptide similarly affected cells indicating signaling through protease activated receptor 1. A direct effect on fibrinolysis was shown by observations of increased plasminogen binding to HUVEC that was inhibited by anti-p11 antibody, and activation of plasminogen by tissue plasminogen activator. These data suggest a novel link between thrombin and fibrinolysis, and other processes that may involve A2, such as cytomegalovirus infection.