Signaling through the rapamycin-sensitive mammalian target of rapamycin-p70 S6 kinase (p70 S6K) pathway was examined in human preadipocytes in primary culture. The inhibitory effect of rapamycin on insulin-induced differentiation of preadipose cell lines has been attributed to a blockade of clonal expansion. We demonstrate that rapamycin inhibits the differentiation of human preadipocytes in primary culture, even though these cells do not undergo clonal expansion. Glycerol-3-phosphate-dehydrogenase (GPDH) activity, an indicator of differentiation, was reduced in omental and subcutaneous preadipocytes to 17 +/- 10% (mean +/- 95% confidence limits, n = 10) of standard differentiation. Our data suggest rapamycin-sensitive pathways operate independently of clonal expansion. Our second objective addressed a novel route of p70 S6K activation. We have detected the presence of TSH receptor in human preadipocytes, and have shown it activates PKB and p70 S6K in a wortmannin-sensitive manner. A model depicting the convergence of insulin and TSH signaling in human preadipocytes is proposed.

Mechanosensitive (MS) channels are those whose open probability (P open) changes in response to mechanical stimulation. Principally, there are two types of mechanical effects on the channel activity, stretch-activation and stretch-inactivation. Mechano-responses can be detected with patch-clamp recordings in cells from bacteria to human, indicating the ubiquitous distribution of MS channels. Mechanosensitivity has recently been shown to be associated with a variety of unrelated channel types, suggesting that susceptibility to membrane tension could be an inherent property of many integral membrane proteins. In this thesis, I tested the hypothesis that an arbitrarily chosen channel with no known mechanotransducer function could be mechanosusceptible under conditions when (1) studied by patch-clamp recordings and (2) heterologously expressed where the protective machinery of its native environment is absent. In order to test this hypothesis, a simple well characterized voltage-dependent K+ channel, Shaker-Inactivation-Removed (Shaker-IR) was expressed in Xenopus oocytes in which the endogenous MS channels were blocked by Gd3+ (100 muM GdCl3) to facilitate the observation of mechano-response of Shaker-IR. Characteristic conductance-voltage (G-V) curves were obtained for Shaker-IR by two-electrode voltage clamp recordings, although Gd3+ shifted the voltage gating by 23 mV. Shaker-IR currents (ISh) were studied in cell-attached and inside-out excised patches with or without mechanical stimulations. In macropatches, various mechanical effects on Ish were recorded at different activation levels (i.e. at different membrane potentials). At the foot of the G-V curves where Popen was low, stretch-activation dominated the MS Shaker-IR channel activity. At the intermediate range of the curves, only minor stretch effects were detectable. At the top of the curves where Popen was high, sustained stretch frequently caused stretch-inactivation. In small patches, stretch-activation and stretch-inactivation were evident as single channel events. Single channel recordings revealed that at a given membrane potential, Popen changed steeply with the applied pressure, in a dose-dependent manner. As in macro-patch recordings, both stretch-activation and stretch-inactivation were induced in the same patch, depending on the pre-stretch Popen. Before reaching a conclusion that the observed mechanical effects were indicative of the mechanosusceptibility of a voltage-dependent channel, it was necessary to rule out the possibility that these effects might be caused by stretch-induced changes in membrane area or by "breakthrough currents" from the oocytes' endogenous MS channels. These possibilities were excluded by the following arguments: (1) Mechanical stimuli produced no apparent increase in capacitance of the patched-membrane, while the increase of the channel activity upon stretch was often one to two orders of magnitude. (2) Stretch-inactivation (not expected from increased area of the recording patch) occurred and could be observed in the same patch that showed stretch-activation. (3) Both the activation and inactivation could be elicited by either suction or blowing. (4) None of these responses were observed in no-Shaker-IR control oocytes. (5) The effects of mechanical stretch depended on the membrane potential, i.e. on the pre-stretch Popen of the Shaker-IR channel. In conclusion, membrane stretch can interfere with the voltage gating of Shaker-IR, consistent with the assumption that mechanosusceptibility may be an inherent property of many integral membrane proteins. Since mechanosusceptibility is easy to demonstrate, it might play a role in the physiology of cells by (a) modifying the activity of ion channels and (b) creating a need for mechanoprotection for channels when they are not used to monitor bilayer tension.

CD1 molecules are non-polymorphic glycoproteins distantly related to major histocompatibility complex (MHC) molecules and represent a third lineage of antigen-presenting molecules in the immune system. An unusual subset of CD1d restricted T cells in mice called NK T cells, express an invariant Valpha14-Jalpha281 T cell receptor (TCR)alpha rearrangement. NK T cells have been shown to play a pivotal role in the regulation of the immune response, the development of autoimmunity and in tumour rejection. By immunizing mice with CD1d expressing transfectant cells, a panel of CD1d restricted, directly reactive T cell clones was generated. Functionally similar to NK T cells, these T cell clones secreted both Type1 (IFN-gamma) and Type 2 (IL-4, IL-10) cytokines upon stimulation. In contrast, detailed TCR analysis revealed that a diverse repertoire of T cells could recognize CD1d. T cells expressing Valpha10, -11, -15 and -17 and having non-germline-encoded nucleotides resulting in diverse V-J junctions were identified. Three clones expressed the invariant Valpha14-Jalpha281 TCR but were functionally indistinguishable from the clones expressing diverse TCRs. Murine gammadelta T cells were examined for similar reactivity to CD1d, but specific recognition of CD1d could not be demonstrated. These data establish that the universe of TCRs capable of direct recognition of CD1d is more diverse than was previously appreciated, indicating the flexibility of the adaptive immune system in response to CD1d mediated processes. (Abstract shortened by UMI.)

The relationship between the sensitivity of microtubules (Mts) to MeHg-induced disassembly and the extent apoptosis was examined in undifferentiated and neuronally differentiated P19 cells. The extent of Mt disassembly was examined qualitatively by immunofluorescence microscopy and quantitatively by polymer and soluble protein extractions, followed by dot blotting or by SDS-PAGE and western blotting. In both undifferentiated and neuronally differentiated P19 cells MeHg treatments disassembled Mts in a time- and dose-dependent manner, as assessed by immunofluorescence microscopy. However, when the extent of Mt disassembly was assessed by quantitative dot blotting or by SDS-PAGE followed by western blotting there was no change observed in the amount of tubulin in the polymer and soluble fractions between control and MeHg-treated samples. These data suggest that MeHg treatments disassemble Mts into a form which is not extractable as soluble protein. MeHg-induced apoptosis was assessed using a combination of assays including single cell gel electrophoresis (Comet assay), TUNEL, conventional agarose gel electrophoresis, and pulsed field gel electrophoresis. (Abstract shortened by UMI.)

All-trans retinoic acid (tRA) and 8-bromo-adenosine-3 ':5'-cyclic monophosphate (cAMP) have been shown to inhibit proliferation, induce cell differentiation and reverse malignant phenotype of a variety of neuroblastoma cell types in vitro. In this thesis, we chose IMR-32 human neuroblastoma cell line because it exhibits two of the main defects found in neuroblastoma: N-myc gene amplification and defective high affinity NGF receptor Trk-A. Treated IMR-32 cells were analyzed for alterations in cell growth and cell morphology. In the course of the study we also investigated the effect of the two differentiation inducers on the expression and distribution of neurofilament (NF) proteins in the IMR-32 cell line. Diagnosis of various other cancers by studying the structural changes at the molecular level using Fourier Transform Infrared Spectroscopy (FTIR) is well established. The main objective of this thesis work was to determine whether diagnostic bands in FTIR spectrum reflect the mitotic ability of IMR-32 cells and whether treatment with tRA and cAMP induces spectral changes characteristic of normal cells. (Abstract shortened by UMI.)

In the absence of hormone, the glucocorticoid receptor (GR) resides in the cytoplasm existing in an inactive form complexed to heat shock proteins and immunophilins. Upon ligand addition, GR dissociates from the protein complex and transforms into an active free form that rapidly and completely translocates to the nucleus to regulate transcription of specific genes. Upon ligand withdrawal, GR is re-packaged into its multiprotein heterocomplex and is slowly redistributed to the cytoplasm. In this study an indirect immunofluorescence assay was employed to characterize the subcellular localization of a series of GR mutants effectively truncating, deleting or mutating specific regions of the receptor N-terminus. In the first part of my study, I delimited a specific region of GR (amino acids 100--200 of the N-terminus) necessary for both its initial cytoplasmic localization prior to ligand addition and return to the cytoplasm upon subsequent hormone withdrawal. Nuclear import of liganded GR is mediated through a well characterized sequence called NL1, which is adjacent to the receptor DNA binding domain and a second uncharacterized motif NL2, located in the ligand binding domain. Not much has been known about the NL2 mediated nuclear import of GR. The second part of my study was devoted to further characterizing the kinetics of an NL1 deficient GR mutant. (Abstract shortened by UMI.)

The overall objective of this research was to characterize the differences in the metabolic control of oxidative phosphorylation in drug sensitive and drug resistant leukemic cells. Anti-cancer agents may function to promote tumor cell death in a number of ways, however acquired or inherent drug resistance is a major problem in successful cancer treatment. The role of mitochondria in apoptosis has received a great deal of attention from a variety of biochemical perspectives in the past few years. Based on evidence that mitochondrial metabolism is altered during apoptotic processes, this research built on the hypothesis that an alteration in mitochondrial energy metabolism which decreases the production of reactive oxygen species plays a significant role in the protection of tumor cells from cytotoxic therapies. Two pairs of sensitive and resistant leukemic cell lines were studied: the mouse L1210 cell line and its drug resistant L1210/DDP subline, as well as the human HL60 cell line and the HL60/MDR resistant cell line. (Abstract shortened by UMI.)

Inhaled nitric oxide (NO) is frequently administered in combination with hyperoxic gas mixtures to effect pulmonary vasodilation in patients with pulmonary hypertension. NO was recently shown to have a protective effect against the injurious consequences of prolonged hyperoxia. The present study investigated the possibility that this protective effect may be attributable to the ability of NO to block apoptosis. Rats exposed to 100% O2 for 60 h developed severe lung injury consisting of pronounced vascular leak and alveolar apoptosis, as inferred from the presence of DNA fragmentation (positive ISEL staining and DNA ladders in agarose gels), and a decrease in constitutive pro-caspase-3 levels. However, the inclusion of NO (20 ppm) in the hyperoxic gas mixture significantly attenuated both the vascular leak and apoptosis. NO reversed the hyperoxia-associated changes in the activity of the redox-sensitive transcription factors NF-kappaB, AP-1 and Sp1 after 24 h. This protective effect was accompanied by a decrease in the level of the proinflammatory protein ICAM-1 and an increase in the cellular antioxidant glutathione. This study shows for the first time that NO can protect against both hyperoxia-induced apoptosis and vascular leak. The data suggest that the protection may occur at the transcriptional and caspase activation level.

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi) resulting from activation of a pH i regulatory transporter. As pHi regulation had not been studied in a mammalian model, I investigated pHi regulation in the mouse egg during meiotic maturation and egg activation. Steady-state pH i was measured using the pHi sensitive fluorophore SNARF-1-AM in germinal vesicle (GV) oocytes, ovulated eggs, and zygotes. No sustained changes in pHi occurred after germinal vesicle breakdown (GVBD), fertilization or during parthenogenetic egg activation. HCO3-/Cl- exchanger activity was measured in unfertilized eggs and zygotes. Zygotes exhibited a marked intracellular alkalinization and Cl- efflux upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. HCO3-/Cl - exchanger activity was upregulated following in vitro fertilization (IVF) becoming maximal after 79 h. Activation of HCO3- /Cl- exchanger activity appeared to occur by activation of existing, inactive exchangers upregulation of activity was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. HCO3-/Cl- exchanger upregulation was also independent of PKC and cAMP-dependent pathways. Using cycloheximide-activated eggs, HCO3- /Cl- exchanger activation was independent of the repetitive Ca2+i transients. HCO3-/Cl- exchanger activity, measured during the cell cycle, was robust in GV eggs, becoming downregulated during meiotic maturation. Low HCO3- /Cl- exchanger activity was a feature of meiotic metaphase only, as activity was not downregulated during metaphase of the first cell cycle. HCO3-/Cl- exchanger upregulation was dependent on an intact metaphase II spindle and could be blocked by the phosphatase inhibitor okadaic acid following Sr 2+ activation. Finally, HCO3-/Cl - exchanger activity could be activated in unfertilized eggs by the MEK inhibitor U0126. This suggests that HCO3- /Cl- exchanger activity is upregulated at fertilization in the mouse by a cell cycle-dependent mechanism that may involve the MAPK pathway.

Mouse zygotes have been shown to recover from an imposed increase in volume, a process known as a regulatory volume decrease or RVD, via separate but functionally coupled Cl- and K+ channels. The objectives of this thesis were to further determine some of the characteristics of these Cl- and K+ channels involved in RVD in the mouse zygote. It was proposed that the Cl - channel in mouse zygotes was a swelling-activated Cl -/organic osmolyte channel like that found in many other cells. A method developed by Passantes-Morales et al. (1993) was used, in which a large extracellular concentration of one of a number of organic osmolytes was added to the experimental media, and the ability of the zygote to perform RVD in the presence of each was determined. A number of organic compounds demonstrated an inhibition of RVD, and thus, identified a number of organic compounds which might function as organic osmolytes in embryos. To directly test whether swelling of zygotes increased their permeability to organic osmolytes; the permeability in swelled zygotes was compared to that in non-swelled zygotes, using 3H glycine. (Abstract shortened by UMI.)