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CELLULAR REQUIREMENTS FOR MBLAC1 EXPRESSION AS ASSESSED IN MBLAC1-/- MOUSE EMBRYONIC FIBROBLASTS
Title
CELLULAR REQUIREMENTS FOR MBLAC1 EXPRESSION AS ASSESSED IN MBLAC1-/- MOUSE EMBRYONIC FIBROBLASTS.
Creator
McGovern, Samantha, Blakely, Randy, Florida Atlantic University, Department of Biomedical Science, Charles E. Schmidt College of Science
Abstract/Description
The majority of research on drug addiction centers on dopamine (DA)- driven synaptic plasticities and how these changes ultimately lead to compulsive drug seeking. However, growing evidence supports a role of glial factors in various steps that lead to drug abuse and addiction. In this regard, significant evidence implicates glial glutamate (Glu) transporters (GLT-1) and cystine/Glu exchangers (xCT) in determining synaptic and extrasynaptic levels of Glu that support the acute and chronic...
Show moreThe majority of research on drug addiction centers on dopamine (DA)- driven synaptic plasticities and how these changes ultimately lead to compulsive drug seeking. However, growing evidence supports a role of glial factors in various steps that lead to drug abuse and addiction. In this regard, significant evidence implicates glial glutamate (Glu) transporters (GLT-1) and cystine/Glu exchangers (xCT) in determining synaptic and extrasynaptic levels of Glu that support the acute and chronic actions of drugs of abuse. -lactam antibiotics have been found in rodent models to upregulate CNS GLT-1 and xCT and thereby contribute to reinstatement after chronic drug exposure and withdrawal. Previously, the Blakely lab identified a glial expressing gene, swip-10, in Caenorhabditis elegans, whose deletion results in the hyperdominergic phenotype Swimming-Induced Paralysis (Swip), supported by Glu signalingdependent DA neuron hyperexcitability that ultimately drives oxidative stress and DA neuron degeneration. Both SWIP-10 and its putative mammalian ortholog MBLAC1 possess a highly conserved metallo -lactamase domain, and MBLAC1 has been found to bind the Glu modulating, b-lactam antibiotic ceftriaxone (Cef). Indeed, immunodepletion studies indicate that MBLAC1 may be the major highaffinity Cef-binding protein in the brain, leading to the hypothesis that MBLAC1 has a Glu modulatory role(s). Recently a functional role of MBLAC1 been proposed, involving activity as a 3’ exonuclease that processes polyA- mRNAs, including RNAs encoding cell replication-dependent histones. How this role, or others, may support the actions of MBLAC1 in the brain and the non-microbial actions of Cef to extracellular Glu homeostasis, is unclear. Recently, the Blakely lab generated Mblac1-/- mice as a tool to investigate these issues. The following work investigated the requirements of MBLAC1 in growth and the actions of Cef in mouse embryonic fibroblasts (MEFs) cultured from either Mblac1+/+ and Mblac1-/- mice. The presented data suggested that Mblac1-/- MEFs display attenuated growth and cell proliferation relative to Mblac1+/+ MEFs. For the first time, the in vitro protective actions of Cef against oxidative stress is shown to be dependent on MBLAC1. The following studies presented contribute to a definition of the role of MBLAC1 and as a Cef binding protein in native preparations, with findings that can drive models for the role of MBLAC1 in the CNS.
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Date Issued
2019
PURL
http://purl.flvc.org/fau/fd/FA00013395
Subject Headings
Drug addiction--Research, Amino Acid Transport System X-AG, Mice, Fibroblasts
Format
Document (PDF)
Developmental and Protective Mechanisms of the Ocular Lens.
Title
Developmental and Protective Mechanisms of the Ocular Lens.
Creator
Chauss, Daniel C., Kantorow, Marc, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
The vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of...
Show moreThe vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of visual impairment worldwide. The only treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms that regulate the development and lifelong protection of the lens is critical toward the development of future therapies to prevent or treat cataract. To determine those molecules and mechanisms that may be important for these lens requirements we employed high-throughput RNA sequencing of microdissected differentiation statespecific lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified differentiation state-specific molecules that regulate mitochondrial populations between lens epithelial cells that require the maintenance of a functional population of mitochondria and lens fiber cells that must eliminate their mitochondria for their maturation. In addition, we discovered a novel mechanism for how lens epithelial cells clear apoptotic cell debris that could arise from damage to the lens and found that UVlight likely compromises this system. Moreover, the data herein provide a framework to determine novel lens cell differentiation state-specific mechanisms. Future studies are required to determine the requirements of the identified molecules and mechanisms during lens development, lens defense against damage, and cataract formation.
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Date Issued
2016
PURL
http://purl.flvc.org/fau/fd/FA00004577
Subject Headings
Eye--Diseases--Etiology., Cell differentiation., Cellular signal transduction., Protein folding., Mitochondrial pathology., Cellular control mechanisms., Apoptosis., Oxidative stress--Prevention.
Format
Document (PDF)
EFFECTS OF SPECIFIC PfEMP1 LIGATION INTERACTIONS WITH ICAM-1, INTEGRIN αVβ3, AND CD36 ON MONOCYTES IN AN IN VITRO MALARIANAÏVE HOST MODEL
Title
EFFECTS OF SPECIFIC PfEMP1 LIGATION INTERACTIONS WITH ICAM-1, INTEGRIN αVβ3, AND CD36 ON MONOCYTES IN AN IN VITRO MALARIANAÏVE HOST MODEL.
Creator
Merritt, Jordan, Oleinikov, Andrew, Florida Atlantic University, Department of Biomedical Science, Charles E. Schmidt College of Science
Abstract/Description
Malaria is a severe global health problem that causes approximately 435,000 deaths per year. Any non-immune individual traveling to malaria endemic regions can be affected too, including humanitarian volunteers, travelers, and US troops. Under physiological conditions, damaged or malaria-infected RBCs would be removed within the spleen, but Plasmodium falciparum infected RBCs (iRBCs) sequester to microvascular endothelial cells to avoid entering the spleen. Adhesion interactions and parasite...
Show moreMalaria is a severe global health problem that causes approximately 435,000 deaths per year. Any non-immune individual traveling to malaria endemic regions can be affected too, including humanitarian volunteers, travelers, and US troops. Under physiological conditions, damaged or malaria-infected RBCs would be removed within the spleen, but Plasmodium falciparum infected RBCs (iRBCs) sequester to microvascular endothelial cells to avoid entering the spleen. Adhesion interactions and parasite sequestration to endothelial cells are mediated by Plasmodium falciparum erythrocyte membrane protein 1 family (PfEMP1) proteins expressed on the iRBC’s surface. The PfEMP1 proteins bind to existing endothelial cell surface receptors that already serve primary functions, including ICAM-1, integrin αVβ3, and CD36. Traditionally, these receptors are explored in the context of endothelial cell sequestration, but this project examines the consequence of receptor::PfEMP1 interaction on immune cells, namely monocyte-like THP-1 cells.
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Date Issued
2019
PURL
http://purl.flvc.org/fau/fd/FA00013398
Subject Headings
Malaria, Plasmodium falciparum, Integrins, Monocytes, Intercellular Adhesion Molecule-1, CD36 Antigens, Adhesiveness
Format
Document (PDF)
In search of MMP specific inhibitors
Title
In search of MMP specific inhibitors: protein engineering of TIMPs.
Creator
Bahudhanapati, Harinathachari., Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of...
Show moreThe tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of the active site) and unprimed (left side) regions of the active site. Substitutions for Thr2 of N-TIMP- 1 strongly influence MMP selectivity. In this study we found that Arg and Gly, which generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the NTIMP-1 mutant with AB loop of TIMP-2, it produced a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and MMP-9, respectively. The Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 uM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily. In collaboration with Dr. Yingnan Zhang at Genentech, we have developed a protocol for the phage display of full-length human TIMP-2 to identify high-affinity selective inhibitors of human MMP-1, a protease that plays a role in cleaving extracellular matrix (ECM) components, connective tissue remodeling during development, angiogenesis, and apoptosis. We have generated a library containing 2x1010 variants of TIMP-2 randomized at residues 2-6 (L1), at residues 34-40 (L2) and 67-70 (L3)., The L1 library yielded a positive signal for MMP-1 binding. Clones from the L1 library, designated TM1, TM8, TM13, and TM14, were isolated after 5 rounds of selection on immobilized MMP-1 and MMP-3 and found to show a greater selectivity for MMP-1 relative to MMP-3. TM8, which has Ser2 to Asp and Ser4 to Ala substitutions, showed the greatest apparent selectivity of 10-fold toward MMP-1 compared to MMP-3. The various mutations identified by phage display were introduced into recombinant Nterminal TIMP-2 and the variants characterized as inhibitors of an array of MMP catalytic domains. The TM8-based mutant showed pronounced selectivity (> 1000-fold for MMP-1 vs. MMP-3) and may be a step towards the generation of MMP-1-specific inhibitors. Molecular modeling was used to rationalize the structural basis of MMP selectivity in the mutants.
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Date Issued
2009
PURL
http://purl.flvc.org/FAU/221942
Subject Headings
Metalloproteinases, Inhibitors, Apoptosis, Extracellular matrix proteins, Proteolytic enzymes
Format
Document (PDF)
New insights into the neuromodulatory role and potential action site of taurine in retinal neurons
Title
New insights into the neuromodulatory role and potential action site of taurine in retinal neurons.
Creator
Bulley, Simon, Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
Taurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study...
Show moreTaurine is the second most abundant amino acid in the CNS after glutamate and its functions have been found largely related to intracellular calcium ([Ca2+]i) modulation, osmoregulation, membrane stabilization, reproduction and immunity. The action of taurine has also been implicated in neurotransmission and neuromodulation though its specific sites of action are not fully understood. Isolated retinal neurons from the larval tiger salamanders (Ambystoma tigrinum) were used as a model to study the neuromodulatory role of taurine in the CNS and to gain insights into its potential sites of action. A combination of techniques was used, including whole-cell patch clamp recording to study taurine's regulation of voltage-gated potassium (K+) and Ca2+ channels and Fluo-4AM Ca2+-imaging to study taurine's regulation of glutamate-induced [Ca2+] I,. Taurine was shown to suppress of glutamate-induced [Ca2+] l, in a dose dependent manner. This suppression was mostly sensitive to the glycine rece ptor antagonist Strychnine but insensitive to any GABA receptor antagonist. The remaining strychnine-insensitive effect was inhibited with the protein kinase A (PKA) inhibitor, PKI, suggesting that there was an additional metabotropic pathway. Moreover, using the protein kinase C (PKC) inhibitor, GF109203X, there was an enhancement in strychnine-insensitive taurine's regulation. Taurine inhibits voltage-gated Ca2+ channels in the retinal neurons and has a dual effect on voltage-gated K+ channels. Taurine causes an increase in K+ current amplitude which is further enhanced with PKI and blocked with GF109203X, suggesting that it is through a PKC-dependent pathway negatively controlled by PKA-dependent pathway., There is a suppression of K+ current by taurine with intracellular application of GF109203X, suggesting that the reduction is through a PKA-dependent pathway. With both PKC and PKA inhibitors there is no longer an enhancement in maximum amplitude but a shift of volt dependence on a hyperpolarizing direction. Taurine's enhancement of K+ current is blocked by the Kv1.3 subtype antagonist Margatoxin, with Kv1.3 accounting for the majority of delayed-rectifier sustained current in bipolar and amacrine cells, as well as 50% of ganglion cells. Interestingly, the enhancement of K+ current by taurine is blocked by 5HT2A antagonist MDL11939, suggesting that activation of PKC is through this metabotropic serotonin receptor subtype. The suppression of voltage-gated Ca2+ channels is reversed with a combination of MDL11939 and the 5HT1A antagonist NAN-190. These results provide the evidence that the natural effect of taurine in the retinal neurons might be dependent on the activation of both 5HT1A and 5HT2A receptors. The high apparent activity of taurine on 5HT receptors could have important implication for the actions of taurine in central brain in which taurine has been known to be beneficial for improving mental health, as well as learning and memory processes.
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Date Issued
2010
PURL
http://purl.flvc.org/FAU/2953206
Subject Headings
Biological transport, Eye, Physiology, Taurine, Physiological effect, Taurine, Therapeutic use, Central nervous system, Physiology
Format
Document (PDF)
Peroxiredoxin 3 and Methionine sulfoxide reductase A are Essential for Lens Cell Viability by Preserving Lens Cell Mitochondrial Function through Repair of Cytochrome c
Title
Peroxiredoxin 3 and Methionine sulfoxide reductase A are Essential for Lens Cell Viability by Preserving Lens Cell Mitochondrial Function through Repair of Cytochrome c.
Creator
Lee, Wanda, Florida Atlantic University, Kantorow, Marc, Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
The central premise of this dissertation is that mitochondrial antioxidant enzymes are essential to lens cell viability by preserving lens cell mitochondria and protecting and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes, Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored. In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization to the lens cell mitochondria, its ability to be...
Show moreThe central premise of this dissertation is that mitochondrial antioxidant enzymes are essential to lens cell viability by preserving lens cell mitochondria and protecting and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes, Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored. In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization to the lens cell mitochondria, its ability to be induced by H20 2-oxidative stress, and speculate how PRDX3 function/sf could affect the lens. We will also examine the reduced levels of MsrA by targeted gene silencing and its effect on reactive oxygen species production and mitochondrial membrane potential in human lens cells to determine its role in mitochondrial function in the lens. Lastly, we will examine the ability of MsrA to repair and restore function to a critical mitochondrial protein, Cytochrome c. The collective evidence strongly indicates that the loss of mitochondrial-specific enzymes, such as PRDX3 and MsrA, are responsible for increased reactive oxygen species levels, decreased mitochondrial membrane potential, protein aggregation and lens cell death, and further indicates that mitochondrial repair, protective, and reducing systems play key roles in the progression of age-related cataract and other agerelated diseases.
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Date Issued
2008
PURL
http://purl.flvc.org/fau/fd/FA00000868
Subject Headings
Genetic regulation, Proteins--Chemical modification, Cellular signal transduction, Eye--Physiology, Mitochondrial pathology
Format
Document (PDF)
Potential therapies and neuroprotective cascades in anoxia tolerant freshwater turtle Trachemys scripta ellegans
Title
Potential therapies and neuroprotective cascades in anoxia tolerant freshwater turtle Trachemys scripta ellegans.
Creator
Nayak, Gauri., Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
Mammalian neurons exhibit extreme sensitivity to oxygen deprivation and undergo rapid and irreversible degeneration when oxygen supply is curtailed. Though several neuroprotective pathways are activated during oxygen deprivation, their analyses are masked by the complex series of pathological events which are triggered simultaneously. Such events can be analyzed in the anoxia tolerant fresh water turtle, which can inherently survive the conditions of oxygen deprivation and post-anoxic...
Show moreMammalian neurons exhibit extreme sensitivity to oxygen deprivation and undergo rapid and irreversible degeneration when oxygen supply is curtailed. Though several neuroprotective pathways are activated during oxygen deprivation, their analyses are masked by the complex series of pathological events which are triggered simultaneously. Such events can be analyzed in the anoxia tolerant fresh water turtle, which can inherently survive the conditions of oxygen deprivation and post-anoxic reoxygenation without brain damage. It is likely in such a model that modulation of a particular molecular pathway is adaptive rather than pathological. The major objective behind this study was to analyze the intracellular signaling pathways mediating the protective effects of adenosine, a potential neuromodulator, and its effect on cell survival by influencing the key prosurvival proteins that prevent apoptosis. In vivo and in vitro studies have shown that adenosine acts as a neuroprotective metabolite and its action can be duplicated or abrogated using specific agonist and antagonists. Stimulating the adenosine receptors using selective A1 receptor agonist N6-cyclopentyladenosine (CPA) activated the presumed prosurvival ERK and P13-K/AKT cascade promoting cell survival, and suppression of the receptor using the selective antagonist DPCPX (8- cyclopentyl-1,3-dipropylxanthine) activated the prodeath JNK and P38 pathways. The complex regulation of the MAPK's/AKT signaling cascades was also analyzed using their specific inhibitors. The inhibiton of the ERK and AKT pathway increased cell death, indicating a prosurvival role, whereas inhibiton of the JNK and p38 pathway increased cell survival in this model. In vitro studies have also shown a high Bcl-2/BAX ratio during anoxia and reoxygenation, indicating a strong resistance to cell death via apoptosis., Silencing of the anti-apoptotic Bcl-2 gene using specific siRNA upregulated levels of prodeath BAX, thus altering the Bcl-2/BAX ratio and elevating cleaved Caspase-3 levels leading to increased cell death. Another promising neuroprotective target which we analyzed was Neuroglobin, which was induced during oxygen crisis and silencing this gene indicated that its plays a major role in modulation of ROS. This study strongly emphasizes the advantages of an alternate animal model in elucidating neuroprotective mechanisms and revealing novel therapeutic targets which could eventually help clinicians to design new stroke therapies based on naturally tolerant organisms.
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Date Issued
2009
PURL
http://purl.flvc.org/FAU/186762
Subject Headings
Turtles, Physiology, Adenosine, Receptors, Cellular signal transduction, Molecular neurobiology, Apoptosis, Research, Cellular control mechanisms
Format
Document (PDF)
Protective Mechanisms of Granulocyte-Colony Stimulating Factor Against Experimental Models of Stroke
Title
Protective Mechanisms of Granulocyte-Colony Stimulating Factor Against Experimental Models of Stroke.
Creator
Menzie-Suderam, Janet, Wu, Jang-Yen, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
Ischemic stroke has a multiplicity of pathophysiological mechanisms. Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exerts a diverse range of neuroprotection against ischemic stroke. Several lines of evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to 10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and 250ng/ml)...
Show moreIschemic stroke has a multiplicity of pathophysiological mechanisms. Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exerts a diverse range of neuroprotection against ischemic stroke. Several lines of evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to 10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and 250ng/ml) for 24 hours. Cell viability, expression of the G-CSF receptor and expression level of CHOP were assessed in vitro. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Rats were subcutaneously injected with G-CSF (n= 15; 50ug/kg body weight) 24 hours post-MCAO for 4 days. Vehicle treated rats were administered 5% dextrose for 1 day (n=4) or 4 days (n=16). Sham-operated rats (n=9) were not subjected to MCAO. Neurological deficit and infarct volume were measured while expression levels of pAKT, Bcl2, Bax, Bak, cleaved caspase-3, GRP78, ATF4, ATF6, p-p38MAPK, pJNK, CHOP and HSP27 were analyzed by western blotting. In vitro G-CSF receptor was expressed on undifferentiated PC12 cell, and an optimal dose of 50 ng/ml G-CSF significantly protected these cells against glutamate-induced cytotoxicity (P < 0.05). G-CSF significantly down-regulated (P < 0.01) the ER stressinduced pro-apoptotic marker CHOP in vitro. In vivo, G-CSF reduced infarct volume to 50% while significantly improved neurological deficit compared to vehicle rats. G-CSF significantly (P < 0.05) up-regulated pro-survival proteins pAKT and Bcl2 while downregulating pro-apoptotic proteins Bax, Bak and cleaved caspase 3 in the ischemic brain. It also significantly (P < 0.05) downregulated the ER intraluminal stress sensor GRP78, proteins of ER stress induced intracellular pathway; ATF4, ATF6, p-p38MAPK, pJNK and the ER stress induced apoptotic marker CHOP, which suggests that ER stress is being ameliorated by G-CSF treatment. G-CSF also reduced the level of HSP27, providing additional evidence of cellular stress reduction. G-CSF treatment increased cell survival by attenuating both general pro-apoptotic proteins and specific effector proteins in the ER stress induced apoptotic pathways. Our data has provided new insight into the anti-apoptotic mechanism of G-CSF, especially as it relates to ER stress induced apoptosis in ischemia.
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Date Issued
2016
PURL
http://purl.flvc.org/fau/fd/FA00004795, http://purl.flvc.org/fau/fd/FA00004795
Subject Headings
Cerebral ischemia--Protection., Apoptosis., Rats as laboratory animals., Cellular signal transduction., Oxidation-reduction reaction.
Format
Document (PDF)
tRNA processing and quality control in bacteria
Title
tRNA processing and quality control in bacteria.
Creator
Alluri, Ravi Kumar, Li, Zhongwei, Charles E. Schmidt College of Science, Department of Biomedical Science
Abstract/Description
In this work, we report that the only exoribonuclease in M. genitalium, RNase R, is able to generate mature 3'-ends. The aminoacyl-acceptor stem, CCA terminus and discriminator residue plays an important role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA, whereas extension of the stem results in the formation of a mature 3’-end. CC residues in CCA terminus and A or G residues at discriminator position are the...
Show moreIn this work, we report that the only exoribonuclease in M. genitalium, RNase R, is able to generate mature 3'-ends. The aminoacyl-acceptor stem, CCA terminus and discriminator residue plays an important role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA, whereas extension of the stem results in the formation of a mature 3’-end. CC residues in CCA terminus and A or G residues at discriminator position are the most preferred residues for precise stopping of RNase R at mature 3’ end. The significance of this works shows that M. genitalium RNase R generates mature tRNA in a single step by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.
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Date Issued
2013
PURL
http://purl.flvc.org/fau/fd/FA00004174
Format
Document (PDF)
Unraveling the molecular mechanism of human polynucleotide phosphorylase (hPNPase) in controlling oxidized RNA
Title
Unraveling the molecular mechanism of human polynucleotide phosphorylase (hPNPase) in controlling oxidized RNA.
Creator
Malla, Sulochan, Li, Zhongwei, Florida Atlantic University, Department of Biomedical Science, Charles E. Schmidt College of Science
Abstract/Description
Oxidation by reactive oxygen species is the major source of RNA damaging insult in living organisms. Increased RNA oxidation has been strongly implicated in a wide range of human diseases; predominantly neurodegeneration. Oxidized RNA should be removed from the cellular system to prevent their deleterious effect to the cells and organisms. In eukaryotic cells, mitochondria are the major intracellular sources of ROS and may cause greater damage to the mitochondrial RNA. In this study, we first...
Show moreOxidation by reactive oxygen species is the major source of RNA damaging insult in living organisms. Increased RNA oxidation has been strongly implicated in a wide range of human diseases; predominantly neurodegeneration. Oxidized RNA should be removed from the cellular system to prevent their deleterious effect to the cells and organisms. In eukaryotic cells, mitochondria are the major intracellular sources of ROS and may cause greater damage to the mitochondrial RNA. In this study, we first investigated the RNA oxidation, by measuring the level of 8-hydroxy-Guanosine (8-oxo-Guo), inside mitochondria and cytoplasm in cultured human cells. We discovered that the mitochondrial 8-oxo-Guo is higher than its cytoplasmic counterparts under both normal growth and oxidative stress condition. Next, we explored the role of human polynucleotide phosphorylase (hPNPase) in controlling RNA oxidation inside mitochondria and cytoplasm. hPNPase binds to oxidized RNA with higher affinity, reduces the 8-oxo-Guo level in total RNA and protects cells against oxidative stress. In this study, the molecular mechanism of hPNPase in 8-oxo-Guo reduction was investigated. First, the effect of hPNPase activities on the 8-oxo-Guo level in mitochondria and cytoplasm was examined. The knockdown of hPNPase increased both the mitochondrial and cytoplasmic 8-oxo-Guo, whereas overexpression had the opposite effect. Second, our study revealed that hSUV3, an RNA helicase that forms a functional complex with hPNPase in mitochondria, was dispensable in reducing 8-oxo-Guo levels.
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Date Issued
2019
PURL
http://purl.flvc.org/fau/fd/FA00013392
Subject Headings
RNA, Reactive Oxygen Species, Mitochondria, Oxidative stress
Format
Document (PDF)