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Scientific Reports May 2018Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for...
Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for bacteria. Here we demonstrate that the development of B. subtilis biofilms is dependent on the use of glutamine or glutamate as a nitrogen source. We show a differential metabolic requirement within the biofilm; while glutamine is necessary for the dividing cells at the edges, the inner cell mass utilizes lactic acid. Our results indicate that biofilm cells preserve a short-term memory of glutamate metabolism. Finally, we establish that drugs that target glutamine and glutamate utilization restrict biofilm development. Overall, our work reveals a spatial regulation of nitrogen and carbon metabolism within the biofilm, which contributes to the fitness of bacterial complex communities. This acquired metabolic division of labor within biofilm can serve as a target for novel anti-biofilm chemotherapies.
Topics: Aminooxyacetic Acid; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Biofilms; Carbon; Diazooxonorleucine; Gene Expression Regulation, Bacterial; Glutamic Acid; Glutamine; Mutant Proteins; Nitrogen; Repressor Proteins
PubMed: 29740028
DOI: 10.1038/s41598-018-25401-z -
The Laryngoscope Feb 2018Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar...
OBJECTIVE
Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro.
METHOD
Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10 M DON.
RESULTS
DON treatment reduced cellular proliferation rate (n = 7, P = 0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P = 0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P = 0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P = 0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption.
CONCLUSION
The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation.
LEVEL OF EVIDENCE
NA. Laryngoscope, 128:E59-E67, 2018.
Topics: Adult; Aged; Antimetabolites, Antineoplastic; Cell Culture Techniques; Cell Proliferation; Cicatrix; Collagen; Diazooxonorleucine; Female; Fibroblasts; Fibrosis; Gene Expression; Glycolysis; Humans; Iatrogenic Disease; Laryngostenosis; Male; Middle Aged; Oxygen Consumption; Real-Time Polymerase Chain Reaction; Tracheal Stenosis; Young Adult
PubMed: 28940431
DOI: 10.1002/lary.26893 -
Scientific Reports Aug 2017Pancreatic cancer is a lethal disease with poor prognosis. Gemcitabine has been the first line systemic treatment for pancreatic cancer. However, the rapid development...
Pancreatic cancer is a lethal disease with poor prognosis. Gemcitabine has been the first line systemic treatment for pancreatic cancer. However, the rapid development of drug resistance has been a major hurdle in gemcitabine therapy leading to unsatisfactory patient outcomes. With the recent renewed understanding of glutamine metabolism involvement in drug resistance and immuno-response, we investigated the anti-tumor effect of a glutamine analog (6-diazo-5-oxo-L-norleucine) as an adjuvant treatment to sensitize chemoresistant pancreatic cancer cells. We demonstrate that disruption of glutamine metabolic pathways improves the efficacy of gemcitabine treatment. Such a disruption induces a cascade of events which impacts glycan biosynthesis through Hexosamine Biosynthesis Pathway (HBP), as well as cellular redox homeostasis, resulting in global changes in protein glycosylation, expression and functional effects. The proteome alterations induced in the resistant cancer cells and the secreted exosomes are intricately associated with the reduction in cell proliferation and the enhancement of cancer cell chemosensitivity. Proteins associated with EGFR signaling, including downstream AKT-mTOR pathways, MAPK pathway, as well as redox enzymes were downregulated in response to disruption of glutamine metabolic pathways.
Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxycytidine; Diazooxonorleucine; Drug Resistance, Neoplasm; Drug Synergism; Glutamine; Humans; Metabolic Networks and Pathways; Oxidation-Reduction; Pancreatic Neoplasms; Proteomics; Gemcitabine
PubMed: 28801576
DOI: 10.1038/s41598-017-08436-6 -
Journal of Medicinal Chemistry Aug 2017Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders...
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.
Topics: Aminocaproates; Animals; Azo Compounds; Blood; Brain; Diazooxonorleucine; Drug Stability; Female; Glutamic Acid; Glutaminase; HIV Infections; Humans; Male; Mice, Inbred C57BL; Neurocognitive Disorders; Nootropic Agents; Prodrugs; Swine; Viral Load
PubMed: 28759224
DOI: 10.1021/acs.jmedchem.7b00966 -
Virology Aug 2017Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that...
Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.
Topics: Alphavirus Infections; Animals; Antiviral Agents; Diazooxonorleucine; Encephalomyelitis; Glutamine; Humans; Interferon-gamma; Male; Mice; Mice, Inbred C57BL; Sindbis Virus
PubMed: 28531865
DOI: 10.1016/j.virol.2017.05.013 -
Protein Science : a Publication of the... Jun 2017Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and...
Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM min and the K was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.
Topics: Catalytic Domain; Crystallography, X-Ray; Diazooxonorleucine; Enzyme Inhibitors; Humans; Protein Structure, Secondary; gamma-Glutamyltransferase
PubMed: 28378915
DOI: 10.1002/pro.3172 -
Journal of Virology Oct 2016Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system...
UNLABELLED
Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease.
IMPORTANCE
Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.
Topics: Alphavirus; Alphavirus Infections; Animals; Cells, Cultured; Central Nervous System; Cytokines; Diazooxonorleucine; Encephalitis; Encephalitis, Viral; Encephalomyelitis; Female; Glutamine; Lymphocytes; Mice; Mice, Inbred C57BL; Paralysis; Sindbis Virus; Virus Replication
PubMed: 27489275
DOI: 10.1128/JVI.01045-16 -
Neurotherapeutics : the Journal of the... Jul 2016Mosquito-borne viruses are important causes of death and long-term neurologic disability due to encephalomyelitis. Studies of mice infected with the alphavirus Sindbis... (Review)
Review
Mosquito-borne viruses are important causes of death and long-term neurologic disability due to encephalomyelitis. Studies of mice infected with the alphavirus Sindbis virus have shown that outcome is dependent on the age and genetic background of the mouse and virulence of the infecting virus. Age-dependent susceptibility reflects the acquisition by neurons of resistance to virus replication and virus-induced cell death with maturation. In mature mice, the populations of neurons most susceptible to infection are in the hippocampus and anterior horn of the spinal cord. Hippocampal infection leads to long-term memory deficits in mice that survive, while motor neuron infection can lead to paralysis and death. Neuronal death is immune-mediated, rather than a direct consequence of virus infection, and associated with entry and differentiation of pathogenic T helper 17 cells in the nervous system. To modulate glutamate excitotoxicity, mice were treated with an N-methyl-D-aspartate receptor antagonist, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists or a glutamine antagonist. The N-methyl-D-aspartate receptor antagonist MK-801 protected hippocampal neurons but not motor neurons, and mice still became paralyzed and died. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonists GYKI-52466 and talampanel protected both hippocampal and motor neurons and prevented paralysis and death. Glutamine antagonist 6-diazo-5-l-norleucine protected hippocampal neurons and improved memory generation in mice surviving infection with an avirulent virus. Surprisingly, in all cases protection was associated with inhibition of the antiviral immune response, reduced entry of inflammatory cells into the central nervous system, and delayed virus clearance, emphasizing the importance of treatment approaches that include prevention of immunopathologic damage.
Topics: Alphavirus Infections; Animals; Benzodiazepines; Cell Death; Diazooxonorleucine; Dizocilpine Maleate; Encephalomyelitis; Hippocampus; Humans; Inflammation; Memory; Mice; Neurons; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sindbis Virus; Survival Analysis; Th17 Cells
PubMed: 27114366
DOI: 10.1007/s13311-016-0434-6 -
Aging Cell Jun 2016Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and...
Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O-GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O-GlcNAcylation in metabolically active rat brain slices by O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non-PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O-GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O-GlcNAcylation through intracerebroventricular injection of 6-diazo-5-oxo-l-norleucine (DON), the inhibitor of glutamine fructose-6-phosphate amidotransferase, suppressed PKA-CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel mechanism that regulates PKA-CREB signaling. Downregulation of O-GlcNAcylation suppresses PKA-CREB signaling and consequently causes learning and memory deficits in AD.
Topics: Acetylglucosamine; Alzheimer Disease; Animals; Brain; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Diazooxonorleucine; Down-Regulation; Glycosylation; HEK293 Cells; HeLa Cells; Humans; Learning; Male; Memory; Memory Disorders; Mice, Inbred C57BL; Phosphorylation; Protein Transport; Rats, Sprague-Dawley; Subcellular Fractions; tau Proteins
PubMed: 26840030
DOI: 10.1111/acel.12449 -
Cell Reports Oct 2015Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic...
Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic reprogramming in T cells regulates T cell activation and differentiation but is not just a consequence of antigen recognition. Although such metabolic reprogramming promotes the differentiation and function of T effector cells, the differentiation of regulatory T cells employs different metabolic reprogramming. Therefore, we hypothesized that inhibition of glycolysis and glutamine metabolism might prevent graft rejection by inhibiting effector generation and function and promoting regulatory T cell generation. We devised an anti-rejection regimen involving the glycolytic inhibitor 2-deoxyglucose (2-DG), the anti-type II diabetes drug metformin, and the inhibitor of glutamine metabolism 6-diazo-5-oxo-L-norleucine (DON). Using this triple-drug regimen, we were able to prevent or delay graft rejection in fully mismatched skin and heart allograft transplantation models.
Topics: Allografts; Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Deoxyglucose; Diazooxonorleucine; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Glutamine; Glycolysis; Graft Rejection; Heart Transplantation; Metformin; Mice; Mice, Inbred BALB C; Mice, Transgenic; Phosphorylation; T-Lymphocytes, Regulatory
PubMed: 26489460
DOI: 10.1016/j.celrep.2015.09.036