Cellular and Molecular Science

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21755

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    X-RAY FLUORESCENCE FOR TRACKING CELL DIFFERENTIATION
    (2014-03) Lin, Victor; Dimitrijevich, Dan; UHV Technologies, Inc; Di Pasqua, Anthony
    The importance of C, H, N, S, and P as essential components of biomolecules and the nutritional value of Ca, Mg, Na, K, P, and Cl are well known. However, the roles of trace elements such as Cr, Co, Cu, I, F, Fe, Mn, Mo, Se and Zn in stem cells and cell differentiation are not known. Cell signaling (Ca), activity of metalloproteins (Cu, Zn, Fe, Mg, Mo, Se, Mn), enzymes (Mg), metallothionines, superoxide dismutase (Cu, Zn), matrix metalloproteinases (Zn) and glycosyltransferases (Mn) depend on molecules that rely on elements to maintain tissue homeostasis. Since tissue homeostasis depends on cell proliferation and differentiation it is expected that there will be a corresponding change in concentration of elements. The possibility of characteristic element concentrations signatures (ECS) provides rational for studying the role of these elements in cell differentiation. The technology that can accomplish this is X-ray fluorescence (XRF). Purpose (a): To analyze using XRF several breast cancer cell lines as a model for changes in cell phenotype and function. To validate these ECSs and begin to compile a database will facilitate non-destructive tracking of stem cell differentiation in vitro and their applications to regenerative medicine. Methods (b): XRF spectroscopy (Bruker, PicoFox) was used to analyze multiple breast cancer cell lines (e.g. MCF10A, T47D, and MCF7) cultured on special discs to determine their ECSs. The ECSs of cell line cells were validated using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Results (c): After normalization to phosphorous (P) as a control for all 3 cell lines, MCF7 exhibited increased S, K, and Zn levels, while T47D exhibited increased Ca, Fe, Cl and Cu levels. The changes in the T47D cell line, for example, suggest appropriate correlation to the cancer cell's metabolic and functional properties: (i) higher Ca levels seen in micro-calcification, (ii) higher Fe levels likely due to elevated mitochondrial activity, (iii) higher Cl levels due to increased ion transport and (iv) higher Cu levels related to increased proliferation, possible interaction with Cu-superoxide dismutase, and involvement in growth factor signaling. Conclusions (d): Thus, preliminary testing shows the power of XRF technology to distinguish different cell phenotypes. This provides support for our hypothesis that XRF measurements of ECSs can distinguish cell phenotypes and will be useful tool for future characterization of adult stem cells and their differentiation progeny.
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    METHAMPHETAMINE (METH) REGULATES ASTROCYTE EXCITATORY AMINO ACID TRANSPORTER-2 (EAAT-2) VIA ACTIVATION OF TRACE AMINE ASSOCIATED RECEPTOR (TAAR1) AND DOWNSTREAM CAMP SIGNALING
    (2014-03) Cisneros, Irma E.; Ghorpade, Anuja
    Methamphetamine (METH) abuse has prevailed as a drug epidemic within the United States and worldwide with an estimated 27.4 million users.Short-term side effect of METH abuse include a heightened libido and impaired judgment increasing the users chances of contracting Human immunodeficiency virus-1 (HIV-1). HIV-1 associated dementia (HAD), is the most severe manifestation of HIV-1-associated neurocognitive disorders and is an important neurological complication of HIV-1 infection characterized by cognitive, behavioral and motor dysfunction. Long-term METH users share characterized neurocognitive defects and disorders as HIV-1 infected individuals.Molecular outcomes of METH/HIV-1-induced neurotoxicity include Excitotoxicity, oxidative stress, glial cell activation, inflammation, and hyperthermia. Astrogliosis is a key pathological feature of METH exposure and HAD, however, the molecular mechanisms remain unclear. The current studies investigate METH-induced TAAR1 activation and will uncover molecular mechanisms associated with glutamate transporter (EAAT-2) dysregulation. Purpose (a): Glutamate is an excitatory neurotransmitter that is highly regulated in the central nervous system (CNS). High concentrations of extracellular glutamate result in excitotoxicity and can exacerbate neurodegenerative disorders, including human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). Additionally, drugs of abuse such as methamphetamine (METH) can increase the severity of excitotoxicity and can accelerate HAND. Excitatory amino acid transporter-2 (EAAT-2) is responsible for approximately 90% of extracellular glutamate uptake from the synapse and is primarily localized in astrocytes. Dysregulation of EAAT-2 leads to astrocytes decreased ability to clear glutamate. Methods (b): It is established that METH leads to excitotoxicity in neurons, however, in astrocytes the molecular mechanisms resulting in METH-mediated EAAT-2 dysregulation are unclear. Previously we showed that HIV-1ADA, METH and transient hyperthermia regulates localization and expression of astrocyte trace amine associated receptor 1 (TAAR1). Results (c): Our data shows METH-induced activation of astrocyte TAAR1 increases intracellular cAMP levels in astrocytes that is significantly decreased in siTAAR1-transfected astrocytes. Further, METH treatment downregulates EAAT-2 mRNA levels. We propose downstream cAMP signaling pathways of METH-induced astrocyte TAAR1 activation result in EAAT-2 dysregulation. Conclusions (d): The results of this study will uncover novel molecular mechanism of METH-induced astrocyte TAAR1 activation and the downstream effects of cAMP signaling on astrocyte EAAT-2 levels in the context of HAND.