Activation of endoplasmic reticulum (ER) stress, notably the unfolded protein response (UPR) and ER-associated degradation (ERAD), contributes to injury in certain renal glomerular diseases. Protein-tyrosine phosphatase 1B (PTP1B) was previously shown to enhance the IRE1α (inositol requiring-1α) branch of the UPR. We propose that PTP1B is an important modulator of complement-mediated glomerular injury, acting via regulation of IRE1α signaling, including c-Jun N-terminal kinase activation and ERAD. To test this hypothesis, we employed PTP1B-deficient mouse embryonic fibroblasts (MEF) and glomerular epithelial cells (GEC) in which PTP1B activity was blocked using a dominant negative cDNA or chemical inhibitor. Complement was activated by incubating cells with antibody, followed by normal human serum, or decomplemented serum in controls. We show that the complement-mediated decrease in the degradation of the ERAD reporter, CD3δ, was attenuated in PTP1B deficient MEF and GEC, implicating PTP1B as a mediator of ERAD. Surprisingly, overexpression of PTP1B produced an effect similar to PTP1B deficiency on ERAD functionality in complement-stimulated GEC. This result suggests that endogenous levels of PTP1B are tightly regulated, and both overexpression and inhibition of this protein are detrimental to ERAD functionality. Complement-mediated cytotoxicity was reduced after PTP1B overexpression, and there was a tendency toward reduced complement cytotoxicity after inhibition of PTP1B. Moreover, we demonstrated that PTP1B knockout mice with anti-GBM nephritis have decreased proteinuria compared to wild type littermates. This protective effect of PTP1B deficiency correlates with the reduced complement-mediated cell death observed in MEF, and suggests that in PTP1B deficient mice, there is reduced proapoptotic signaling in the glomerulus. In conclusion, the results of the present study demonstrate a novel relationship between PTP1B and ERAD. Additionally, the cytoprotective effect of PTP1B deletion in MEF and in anti-GBM nephritis suggests that PTP1B may potentially be a therapeutic target in complement-mediated diseases. / L'activation du stress du réticulum endoplasmique (ER), notamment la réponse au stress des protéines mal repliées (UPR) et la dégradation associée au réticulum endoplasmique (ERAD), engendre des lésions dans certaines maladies glomérulaires rénales. Il a déjà été prouvé que la Protein-tyrosine phosphatase 1B (PTP1B) régule positivement la branche IRE1α (inositol requiring-1α) d'UPR. Nous proposons que PTP1B est un modulateur important de la lésion glomérulaire provoquée par l'activation du système du complément, aidé par la régulation de la signalisation de IRE1α, incluant l'activation de c-Jun N-terminal kinase et ERAD. Pour tester cette hypothèse, nous avons utilisé des cellules fibroblastes de souris embryonnaires (MEF) déficientes en PTP1B et des cellules épithéliales glomérulaires (GEC) dans lesquelles l'activité de PTP1B a été bloquée en utilisant un ADNc dominant négatif ou un inhibiteur chimique. Le complément est activé par l'incubation des cellules avec l'anticorps anti-GEC, suivi par une incubation avec du sérum humain normal, ou du sérum décomplémenté chez les cellules témoins. Nous montrons que la diminution de la dégradation du gène rapporteur de ERAD, CD3δ, induite par le complément, a été atténuée dans les MEF et les GEC déficientes en PTP1B, impliquant que PTP1B est un médiateur de ERAD. Étonnamment, la surexpression de PTP1B a produit un effet similaire à une déficience de PTP1B sur la fonctionnalité de ERAD dans les GEC qui sont stimulées par le complément. Ce résultat suggère que les niveaux endogènes de PTP1B sont strictement réglementés, et que la surexpression ainsi que l'inhibition de cette protéine sont nuisibles à la bonne fonctionnalité de ERAD. La cytotoxicité médiée par le complément a été réduite après la surexpression de PTP1B, et on a remarqué une tendance à une diminution de la cytotoxicité du complément après l'inhibition de PTP1B. De plus, nous avons démontré que les souris déficientes en PTP1B à qui on a induit la maladie des anticorps anti-membrane basale glomérulaire (anti-GBM) ont une protéinurie moindre par rapport aux souris témoins. Cet effet protecteur de la déficience en PTP1B est en corrélation avec une diminution de la mort cellulaire médiée par le complément observée dans les MEF, et suggère que les souris déficientes en PTP1B ont une réduction de la signalisation pro-apoptotique dans le glomérule. En conclusion, les résultats de la présente étude montrent une relation originale entre PTP1B et ERAD. En outre, l'effet cytoprotecteur de la suppression de PTP1B dans les MEF et dans la maladie des anticorps anti-GBM suggère que PTP1B peut potentiellement être une cible thérapeutique dans les maladies médiées par le complément.

In vertebrate embryos, the forebrain and eyes are subdivisions of anterior neural tissue. The work here demonstrates a novel regulation of anterior neural development by zebrafish lmo4b. Although these developmental processes have been well studied, it still remains unclear how cells are allotted to and maintained in these subdivided regions. We have identified the zebrafish lmo4b gene and show that it is required to position and maintain proper boundary formation between neural and non-neural ectoderm and by doing so, lmo4b restricts the size of anterior neural tissue. Additionally, lmo4b negatively regulates genes involved in forebrain specification and maintenance such as six3b and rx3, which also promote cell proliferation in anterior neural tissue. Precursors of the peripheral sensory organs (lens, nasal epithelium, inner ear and facial motor neurons) are derived from the placodal primordium that is localized in the boundary region. Therefore, lmo4b may regulate placodal ectoderm development. We also show that lmo4b patterns the eye along the proximodistal axis and is regulated by extracellular signals, Shh and Fgf. Zebrafish lmo4b is initially expressed in the anterior ectoderm at the neural/non-neural boundary, later in the telencephalon, optic vesicles and finally in optic stalk and retinal pigmented epithelium progenitors. With gain- and loss-of-function studies by overexpression assays and morpholino knockdowns, respectively, we are able to integrate lmo4b into an intricate web of previously identified genes and signaling pathways that regulate embryonic forebrain and eye development. lmo4b overexpression produces embryos with relatively normal gross morphology; however, they have reduced, or no eyes, smaller forebrain and craniofacial and fin defects. The lmo4b knockdown phenotype includes expanded anterior neural plate, followed by enlarged forebrain and eyes, abnormal ears and fins. Moreover, closer analyses reveal that lmo4b morphants have increased mitotic cell and total cell nuclei counts in the optic vesicles. We propose that because the initial establishment of the presumptive anterior neural boundary is plastic, lmo4b is an essential modulator in positioning the boundary and consequently controls cell commitment to anterior neural fate and tissue growth. We also propose that lmo4b influences the refining of the compartments within the eye through Shh and Fgf regulation.

This work presents some important results from experimental studies aimed at elucidating the fundamental mechanisms of endothelial cell migration and proliferation. To continuously monitor populations of migrating and proliferating cells, we used video microscopy coupled with a novel computer-automated digital time-lapse recording technique. Migrating cells were identified and their positions at each time instant were obtained using digital image processing. We also developed a modified nearest neighbor tracking algorithm to reconstruct approximations to the cell trajectories. Our experimental studies on the locomotion of bovine pulmonary artery endothelial (BPAE) cells have shown that these cells execute persistent random walks in culture. Cells change their direction of migration either in response to some intracellular signal or because they collide with other cells. Cells slow down as they approach other cells and then turn and move away from each other with increasing speeds. The temporal evolution of population-average speed of locomotion reveals that increases in cell density due to proliferation are immediately accompanied by a decrease in the average cell speed. Markov chain analysis on cell trajectories has shown that the enhanced motility of the BPAE cells cultured with basic fibroblast growth factor (bFGF) seems to be derived not only from fewer visits to the stationary state, but also from a decrease in the waiting time for each visit to the stationary state. BPAE cells execute persistent random walks when they are cultured without or with added bFGF, although the addition of bFGF does make them more motile. An independent set of cell proliferation experiments indicates that bFGF concentrations that increase cell motility also increase cell proliferation rates. A model based on a cellular automaton was developed to describe the proliferation of migrating cells. Our cellular automaton models asynchronous proliferation of cells executing persistent random walks and accounts for changes in the direction of movement when two cells collide. The simulation results reveal that cell motility reduces the adverse effects of contact inhibition on cell proliferation rates. Excellent agreement between model predictions and experimental data was observed indicating that this discrete model can accurately describe the dynamics of populations of migrating and proliferating cells.

Cell culture provides a simple and effective system to study metabolic and cellular processes required for cellular growth in vitro. In this study, we demonstrate that lymphocyte culture can be used as a model system to study the mechanism of insulin resistance (obesity/diabetes) as well as the process of aging of cells from the immune system. Our results indicate that metabolic differences can be observed in lymphocytes from obese subjects and that different degrees of alteration in response can be detected between obese nondiabetic and diabetic subjects. Furthermore different effects of insulin resistance can be reflected faithfully in lymphocyte biochemistry in culture. In isolated lymphocytes from normal weight subjects, G3PDH (an enzyme at the intersection of glycolytic and lipogenic pathways) activity increases in the presence of insulin. Augmented G3PDH activity requires new protein synthesis and involves the inositol triphosphate-diacylglycerol signalling system. However, in obese subjects for whom insulin resistance in vitro can be demonstrated, the extent of insulin stimulation of G3PDH activity is decreased compared to normal weight individuals. These results suggest that G3PDH activity is dependent upon the metabolic state of the subjects from which the cells are obtained. Dietary restriction for obese subjects normalizes insulin and glycerol responses and insulin effects on G3PDH activity. These results demonstrate that the lymphocyte can serve in vitro as a model to reflect organismal metabolism in obesity/diabetes. Alterations of lymphocytes due to aging contribute significantly to changes in immune function. In present study, an artificial aging environment was introduced using whole blood. In this environment, we demonstrate that lymphocytes undergo degeneration and aging processes similar to those observed in vivo. Similar changes in growth capacity, lipid peroxidation, surface antigens on T-cells, and adherent cell suppression were observed in lymphocytes aged in vivo or in vitro. Based on these results, we suggest that blood aged in vitro may provide a simple but effective model to study many aspects of cellular aging in vivo. To further examine this hypothesis and to further understand the aging process, vitamin E was used to examine its effect on in vitro aging. The results from lymphocyte growth capacity assays show that vitamin E provides a protective effect against in vitro aging similar to that observed for in vivo aging. Examination of lymphocytes in culture allows monitoring of biochemical alterations, nutrient requirements, and other parameters in vitro, without effect on the subject. This technique therefore provides an opportunity to examine a wide variety of factors and a system eventually in which mechanisms to prevent or impede diseases can be tested.

Cell adhesion to material surfaces is a fundamental phenomenon in tissue response to implanted devices, and an important consideration in tissue engineering. The first objective of this study was to measure the mechanical adhesiveness characteristics of rabbit articular chondrocytes as a function of seeding time to provide further understanding of the cell adhesion process. The second objective was to quantify the tether formation force and tether stiffness as a function of seeding time. With increasing time of seeding up to 6 hours, chondrocytes exhibited increasing mechanical adhesiveness, tether formation force and tether stiffness, as measured using the cytodetacher and optical tweezers system. Concomitantly, cell contact area and cell height, as measured using inverted microscope and confocal imaging, were found to increase and decrease respectively.

ME31B, a putative RNA helicase belongs to the DEAD box family. A subclass of the DEAD box family is growing around ME31B. The members are DHH1 (S. cerevisiae), ste 13 (S. pombe), and rck/p54 (human and murine). In a conserved region of approximately 390 amino acids, these proteins all share greater than 70% identity with ME31B. The construct pOTS-Nco12-ME31B was used to produce an easily discernible quantity of insoluble ME31B. Following treatment with 8 M urea, ME31B was allowed to renature. Approximately 66% of the ME31B protein remained soluble during these manipulations. This soluble protein was purified on an HPLC hydrophobic column with a purification yield from total protein loaded of 35% soluble ME31B. With this soluble pure protein, I was able to investigate ME31B's ability to hydrolyze ATP. In an ATPase assay, ME31B hydrolyzed ATP in the presence and absence of an RNA substrate.

3$\beta$-Hydroxy-cholest-5-en-7-one (15) has been reported to lower 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in both mouse liver cells and whole animals. In experiments with rats, studies have shown that 15 is rapidly metabolized and excreted from the body. We synthesized 3$\beta$-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (19) with the hope that blocking the sidechain metabolism at C-25 and C26 would allow sufficient quantities to accumulate in vivo and possibly lower serum cholesterol levels. 19 was synthesized via 25,26,26,26,27,27,27-heptafluorocholest-5-en-3$\beta$-ol (3) by employing the reagents chromic anhydride and 3,5-dimethylpyrazole (DMP) in methylene chloride (66% yield; $>$99% pure by NMR). A thorough characterization of 19 and its nonfluorinated counterpart 15 is presented. Several byproducts from the synthesis of 3 and 19 were discovered. Their preliminary characterizations are also reported.

I am interested in understanding a mechanism by which multicellular organisms sense number of cells of the same type. The model selected is the conditioned medium factor secreted by Dictyostelium discoideum. In submerged monolayer culture, Dictyostelium cells can differentiate into prespore and prestalk cells at high cell densities in response to cAMP but this process does not occur at low cell densities. However, cells at low densities will differentiate in medium taken from developing cells starved at a high density. The putative factor in the medium was designated CMF for Conditioned Medium Factor (Mehdy and Firtel 1985). In this report, we show that the CMF activity can be separated into high and low molecular weight fractions. The large conditioned medium factor can be purified to a single 80 kD protein with N- and O-linked glycosylation and has CMF activity at a concentration of $\sim$4 pM (0.3 ng/ml). This 80 kD CMF can undergo size reduction to a $\sim$100-fold more active set of smaller peptides with molecular weight less than 10 kD. Glycosylation is required for the activity of the low molecular weight CMF. Starvation triggers the release of CMF from a precursor pool already present in vegetative cells, and diffusion calculations indicate that the CMF level in the vicinity of a single isolate will not accumulate to the threshold concentration $\sim$0.3 ng/ml. CMF antisense transformants do not aggregate, whereas normal development is restored by the addition of purified 80 kD CMF. These results suggest that CMF is a secreted factor that functions in vivo as an indicator of cell density in starved cells. The developing cells simultaneously secrete CMF and monitor its extracellular level. When a majority of the cells in a given area have starved as indicated by the high level of CMF, aggregation is triggered to ensure the onset of development is synchronized. When present below a threshold concentration, the expression of genes required for early development is blocked or not induced. This factor plays an essential role in the regulatory pathway necessary for cells to obtain the developmental competence to induce prestalk and prespore gene expression in response to cAMP.

Much recent emphasis in vascular biology has focused on endothelial cells which form the inner lining of blood vessels. This unique arterial location exposes these cells directly to mechanical forces resulting from blood flow and the transmission of the pressure wave through a compliant vessel. In this study, the effects of the cyclical expansion and relaxation of the vessel wall on endothelial cell metabolism have been modeled using a uniaxial strain device. A normal range of physiological strains for large arteries was examined. The production rates of prostacyclin (PGI$\sb2$), endothelin, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-type 1 (PAI-1) by endothelial cells were constant over 24 hour periods. The production of both PGI$\sb2$ and endothelin was enhanced by cells exposed to a high level of cyclical strain compared to controls, while t-PA production was unaltered. These results were true for human and bovine endothelial cells. The stimulation of endothelin production was dose dependent with the level of strain while PGI$\sb2$ stimulation required a minimal level of strain before increases over controls were observed. Human endothelial cells subjected to cyclical strain showed elevated production of PAI-1 compared to controls. The possibility that cyclical strain could be used to regulate cell function was investigated. Cyclical strain was applied in an on-off-on manner over a 36 hour period with a 12 hour division between initial application of strain and final reapplication of strain. When the strain was stopped, PGI$\sb2$ production rapidly returned to control levels while endothelin production remained elevated but at a level significantly below the initially stimulated rate. Reapplication of cyclical strain caused a return of the endothelin production rate to a level essentially the same as that during the initial stimulation period. This study initiated a quantitative investigation of cyclical strain effects on endothelial cell production of the mRNA levels of prostaglandin H (PGH) synthase gene, the enzyme of which is involved in production of PGI$\sb2$. Preliminary results using a quantitative reverse transcription-polymerase chain reaction technique suggested that mRNA levels of PGH synthase are not altered in response to cyclical strain.

Initial aspects of the molecular mechanisms of cell-substrate interactions were studied under physiologically relevant shear conditions using a parallel-plate flow chamber. The adhesion process was dissected into the initial events of arrest and stabilization using the methodology developed in this study employing video microscopy coupled to digital image processing on a SPARC 2 workstation. The cell-substrate interactions were studied using human melanoma (i.e. parental (MeWo) and two variants selected for resistance to wheat germ agglutinin cytotoxicity (3s5 and 70w)) and Chinese hamster ovary (CHO) cells expressing different levels of the fibronectin integrin, $\alpha\sb5\beta\sb1$, on their surface. These cells allowed investigation of the roles of transglutaminase (TGase) and the integrin surface receptors in the arrest and stabilization events. TGase inhibitors, monodanyslcadaverine and INO-3178, used in the studies with the melanoma cells showed inhibition of the stabilization event with no measurable effect on the arrest event when adhesion to immobilized fibronectin was investigated under flow conditions. Besides fibronectin, other immobilized proteins were evaluated using the melanoma cells such as laminin, vitronectin, type I and type IV collagen, von Willebrand factor, wheat germ agglutinin (WGA), and Peptite-2000. The results showed the arrest and stabilization events are not necessarily mediated to the same degree by the same molecular components. For example, arrest to WGA was very favorable relative to arrest to fibronectin but stabilization was very poor. Some substrates such as human laminin showed both favorable arrest and stabilization behavior relative to fibronectin. Polyclonal antibodies to the fibronectin and vitronectin integrin receptors on the melanoma cells were used to investigate the role of these integrins in the arrest and stabilization events. It appeared that both receptors are involved to some degree in the arrest and stabilization events to both substrates. The adhesion studies of the CHO cells confirmed these findings that increased fibronectin integrin enhanced arrest and stabilization to both fibronectin and vitronectin. In summary, these results suggest that the stabilization of cells to immobilized proteins is in part attributed to transglutaminase cross-linking integrin receptors on the surface of tumor cells to proteins via lysine-glutamine linkages.