Browsing by Subject "calcium"
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Item Cellular Mechanisms in the Ocular Actions of Endothelin(1996-12-01) White, Karen A.; Yorio, Thomas; Pang, Iok-Hou; Dobbs, RichardWhite, Karen A., Cellular Mechanisms in the Ocular Actions of Endothelin. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 1996, 151 pp., 25 tables, 23 figures, references, 111 titles. Endothelins are a family of regulatory peptides which could have important implications in this regulation of aqueous humor outflow and intraocular pressure (IOP). The objectives of this dissertation were to investigate the cellular mechanism of endothelin (ET) receptor interactions in ocular tissues focusing on their effect on second messengers such as phospholipase C (PLC) and calcium, and their interactions with phospholipase A2 (PLA2) in ciliary muscle cells. The hypothesis was that in human ciliary muscle (HCM) cells, endothelin-1 (ET-1), via the ETA receptor and a pertussis toxin sensitive G-protein, activates PLC, which in turn stimulates calcium mobilization. Independent of this pathway, ET-1 also activates PLA2 and increases the release of prostaglandins. These two pathways provide a cellular second messenger balance that influences ciliary smooth muscle contraction. The current study demonstrated that ET-1 and endothelin-2 (ET-2) stimulate calcium mobilization in HCM cells via an ETA receptor subtype. It appears that the increase in intracellular calcium ([Ca2+]i) is the result of ET coupled to PLC via a pertussis toxin sensitive G-protein. A biphasic calcium response is elicited with ET stimulation consisting of a transient increase in [Ca2+]I which appears to be primarily due to release of intracellular stores, followed by a lower sustained phase which appears to be dependent on the influx of extracellular calcium. Endothelin-1 also appears to stimulate an increase in prostaglandin E2 (PGE2) formation through activation of PLA2. Furthermore, it appears that the effects of ET-1 on PLC and calcium are independent of the ET-1 effects on PGE2 production, such that the ET-1 induced increase in [Ca2+]I are coupled to the PLC signaling pathway, whereas increase in PGE2 production appears to be the result of an ETA receptor coupled PLA2. Whether there are different subtypes of ETA receptors or the receptor is coupled through different G-proteins is uncertain. Endothelin-1 and Big ET-1 immunoreactivity was also observed in both HCM and human nonpigmented ciliary epithelial (HNPE) cells. This is the first time that ET-1 and Big ET-1 immunoreactivity has been detected in the HCM cells, suggesting that these cells have the capability to synthesize both peptides. Furthermore, the increase in ET-1 and Big ET-1 immunoreactivity upon stimulation with TNF-α suggests that cytokines may be important regulators of ET synthesis and release. The findings of this research aid in the understanding of the mechanism of action whereby ETs regulate aqueous humor dynamics and IOP. Through a better understanding of the cellular actions of ET, insight is gained into the development of new ocular selective agents acting at the ET receptor.Item Comparison of DNA Extraction Methods From Bone to be Used With the DNA IQ System on the Maxwell 16 for Human Identification(2008-08-01) Lopez, Kristen; John Planz; Arthur Eisenberg; Joseph WarrenLopez, Kristen M., Comparison of DNA Extraction Methods From Bone to be Used with the DNA IQ System on the Maxwell 16 for Human Identification . Masters of Science (Graduate School of Biomedical Sciences), August, 2008, 52 pp., 11 tables, 15 figures, bibliography, 24 titles. Extraction and purification of DNA from human bones is essential for correctly identifying the remains through DNA analysis. Current DNA extraction methods include a demineralization step, which extracts calcium and phosphate from the bone matrix, inactivation of DNAses, and the removal of Polymerase Chain Reaction (PCR) inhibitors. These methods often use harsh chemicals and may allow for residual DNA to be discarded in various wash steps. To assess the effectiveness of DNA extraction from bone samples, two extraction protocols were compared. The first method included a bone demineralization pretreatment solution of Sodium N-Laurylsarcosinate, 0.5 M EDTA, and Proteinase K (20 mg/ml). The second included a pretreatment using a Bone Incubation Buffer by Promega Corporation, with an addition of Proteinase K (18mg/ml). Various incubation times were included to assess the extraction at different time intervals. All extracted samples were purified with the DNA IQ Reference Sample Kit on the automated Maxwell 16 Instrument (Promega Corp.). Full and partial profiles were obtained from samples extracted with the Bone Incubation pretreatment, regardless of incubation time. Profiles were not observed with the standard demineralization pretreatment when amplified at 28 cycles, with partial profiles present in a few samples when amplified at 32 cycles.Item Methamphetamine Activates Trace Amine Associated Receptor 1 to Regulate Astrocyte Excitatory Amino Acid Transporter-2 via Differential CREB Phosphorylation During HIV-Associated Neurocognitive Disorders(Frontiers Media S.A., 2020-11-25) Cisneros, Irma E.; Ghorpade, Anuja; Borgmann, KathleenMethamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-kappaB, transcription factors activated by cAMP, calcium and IL-1beta. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1beta METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.Item Modulating mitochondrial calcium channels (TRPM2/MCU/NCX) as a therapeutic strategy for neurodegenerative disorders(Frontiers Media S.A., 2023-11-06) Johnson, Gretchen A.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.Efficient cellular communication is essential for the brain to regulate diverse functions like muscle contractions, memory formation and recall, decision-making, and task execution. This communication is facilitated by rapid signaling through electrical and chemical messengers, including voltage-gated ion channels and neurotransmitters. These messengers elicit broad responses by propagating action potentials and mediating synaptic transmission. Calcium influx and efflux are essential for releasing neurotransmitters and regulating synaptic transmission. Mitochondria, which are involved in oxidative phosphorylation, and the energy generation process, also interact with the endoplasmic reticulum to store and regulate cytoplasmic calcium levels. The number, morphology, and distribution of mitochondria in different cell types vary based on energy demands. Mitochondrial damage can cause excess reactive oxygen species (ROS) generation. Mitophagy is a selective process that targets and degrades damaged mitochondria via autophagosome-lysosome fusion. Defects in mitophagy can lead to a buildup of ROS and cell death. Numerous studies have attempted to characterize the relationship between mitochondrial dysfunction and calcium dysregulation in neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic lateral sclerosis, spinocerebellar ataxia, and aging. Interventional strategies to reduce mitochondrial damage and accumulation could serve as a therapeutic target, but further research is needed to unravel this potential. This review offers an overview of calcium signaling related to mitochondria in various neuronal cells. It critically examines recent findings, exploring the potential roles that mitochondrial dysfunction might play in multiple neurodegenerative diseases and aging. Furthermore, the review identifies existing gaps in knowledge to guide the direction of future research.Item Presenilins Modulate Cellular Activity of Ryanodine Receptors(2012-12-01) Payne, Andrew J.; Peter KoulenPayne, Andrew J., Presenilins Modulate Cellular Activity of Ryanodine Receptors. Doctor of Philosophy (Biomedical Sciences), December, 2012, 160 pp., 7 tables, 39 figures, bibliography 241 titles. Ryanodine Receptors (RyRs) are large, endoplasmic reticulum (ER) intracellular calcium channels in excitable cells. RyRs are major cellular mediators of calcium-induced calcium release and crucial regulators of intracellular calcium homeostasis. Disruption of RyR function has been described in pathologies of dysregulated calcium such as Alzheimer’s disease (AD). Presenilins (PS1 and PS2) are ER transmembrane proteins expressed in the central nervous system mediating calcium homeostasis and Notch signaling, and act as the proteolytic core of γ-secretase in amyloid cleavage. Previous single channel electrophysiology studies described a direct interaction between RyR and the N-termini of presenilin 1 (PS1NTF) and presenilin 2 (PS2NTF) that resulted in differential modulation of the RyR open probability and mean Ca2+ current at the RyR single channel level. We herein tested the hypothesis that PS1NTF and PS2NTF functionally modulate RyRs in a physiologically relevant in vitro model resulting in changes to RyR-mediated intracellular calcium release. Confocal microscopy, microfluorimetry, and coimmunoprecipitation studies confirmed a physical interaction between RyRs and PS-NTFs in human neuroblastoma SH-SY5Y cells, an in vitro AD model. Live cell fluorescent calcium imaging was used to quantify the effects of overexpression of PS1NTF or PS2NTF on Ca2+ release from RyR mediated stores. PS1NTF was found to increase RyR gating to the full open state at physiologically normal calcium concentrations. Verifying the previous electrophysiology data, PS2NTF had no effect on RyR at physiological calcium concentrations. Mutagenesis of critical cysteine residues on the PSNTFs was applied to determine the effect of specific structure-function differences between PS1 and PS2 molecules that underlie the isoform specific modulation of RyR calcium release. Mutations to PS2NTF removing disulfide bridging cysteines recapitulated PS1NTF-like regulation of RyR Ca2+ release. Our findings indicate that PS1NTF and PS2NTF bind RyRs differentially. Our results indicate a novel mechanism of intracellular calcium regulation by the PS-RyR interaction and a novel target for the treatment of AD, neurodegenerative disorders, and diseases controlled by RyR and PS functions.Item The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-Dependent Protein Kinase II in Pancreatic β-Cells(1998-06-01) Krueger, Kimberly A.; Richard Easom; Rafael Alvarez; S. Dan DimitrijevichKrueger, Kimberly A., The Phosphorylation of Endogenous Substrates by Calcium/Calmodulin-dependent Protein Kinase II in Pancreatic β-cells. Doctor of Philosophy (Biomedical Sciences), June, 1998, 165 pp., 35 illustrations, references, 259 titles. Increasing evidence supports a physiological role for calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the secretion of insulin from pancreatic β-cell. While it has been previously demonstrated that CaM kinase II is activated by glucose in isolated rat islets implicating this enzyme in the secretion process, its cellular targets are unidentified. Potential candidates would likely exhibit strong binding to the enzyme, an association with the cytoskeleton, or an involvement in the secretion process. Based on these criteria, the following study represents an evaluation, in situ, of two proteins to function as substrates for CaM kinase II. Microtubule-associated protein, MAP-2 is one of the best substrates of CaM kinase II in vitro thought to be involved in secretion process. Synapsin I phosphorylation in the neuron by CaM kinase II is essential for neurotransmitter release. Unique to this study, both proteins were determined to be expressed in clonal mouse β-cells (βTC3) and primary rat islet β-cells. By immunoprecipation, in situ phosphorylation of MAP-2 and synapsin I was induced in permeabilized βTC3 cells within a calcium range shown to activate endogenous CaM kinase II under identical conditions. Two-dimensional tryptic phosphopeptide mapping of both proteins revealed that sites phosphorylated by CaM kinase II in vitro, while distinct from sites phosphorylated by protein kinase A in vitro, were largely homologous to those sites phosphorylated in situ upon incubation of the βTC3 cells with increased free calcium. Immunofluorescence verified expression of both proteins in βTC3 cells and pancreatic slices, however, synapsin I exhibited little co-localization with insulin containing dense core granules as demonstrated by immunogold electron microscopy. These data provide evidence that MAP-2 and synapsin I are phosphorylated by CaM kinase II in the pancreatic β-cell in situ. While the data suggest that synapsin I may not be involved in insulin secretion, an association with other known microvesicles of the β-cell, similarly secreted, may be possible. The phosphorylation of these CaM kinase II substrates may reveal an important intermediate step in the mediation of the glucose response in the pancreatic β-cell.Item Traditional Chinese Medicine and Western Medicine Share Similar Philosophical Approaches to Fight COVID-19(JKL International, 2021-08-01) Zhao, Fangfang; Yang, Zhenhong; Wang, Ningqun; Jin, Kunlin; Luo, YuminThough disciplines in the same field, modern medicine (Western medicine) and traditional medicine (Traditional Chinese medicine, TCM) have been viewed as two distinct and divergent fields of medicine and thus differ greatly in their ways of diagnosing, treating, and preventing disease. In brief, Western medicine is primarily an evidence (laboratory)-based science, whereas TCM is more of a healing art based on the theory of Yin and Yang and the five elements in the human body. Therefore, whether TCM and Western medicine could use similar philosophical approaches to treat disease remains unclear. It is well-known that vitamin D enhances immune function and reduces the spread of some viruses. Indeed, recent evidence shows that the blood calcium level is strongly associated with COVID-19 severity, and vitamin D supplementation has shown favorable effects in viral infections. According to TCM theory, the pathogenesis of COVID-19 is closely associated with cold-dampness, an etiological factor in TCM. Cold-dampness could be attenuated by sun exposure and Wenyang herbs, both of which can restore the vitamin D level in the blood in Western medicine. Therefore, TCM and Western medicine could share similar philosophical methods to fight COVID-19 and understanding their philosophical theories could achieve the maximum benefits for treatment of COVID-19 and other diseases.