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Biochemical Pharmacology Oct 2018Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates...
Glutaminase-1 (GLS1) is a mitochondrial enzyme found in endothelial cells (ECs) that metabolizes glutamine to glutamate and ammonia. Although glutaminolysis modulates the function of human umbilical vein ECs, it is not known whether these findings extend to human ECs beyond the fetal circulation. Furthermore, the molecular mechanism by which GLS1 regulates EC function is not defined. In this study, we show that the absence of glutamine in the culture media or the inhibition of GLS1 activity or expression blocked the proliferation and migration of ECs derived from the human umbilical vein, the human aorta, and the human microvasculature. GLS1 inhibition arrested ECs in the G/G phase of the cell cycle and this was associated with a significant decline in cyclin A expression. Restoration of cyclin A expression via adenoviral-mediated gene transfer improved the proliferative, but not the migratory, response of GLS1-inhibited ECs. Glutamine deprivation or GLS1 inhibition also stimulated the production of reactive oxygen species and this was associated with a marked decline in heme oxygenase-1 (HO-1) expression. GLS1 inhibition also sensitized ECs to the cytotoxic effect of hydrogen peroxide and this was prevented by the overexpression of HO-1. In conclusion, the metabolism of glutamine by GLS1 promotes human EC proliferation, migration, and survival irrespective of the vascular source. While cyclin A contributes to the proliferative action of GLS1, HO-1 mediates its pro-survival effect. These results identify GLS1 as a promising therapeutic target in treating diseases associated with aberrant EC proliferation, migration, and viability.
Topics: Aorta; Benzeneacetamides; Cell Movement; Cell Proliferation; Cell Survival; Cyclin A; Diazooxonorleucine; Endothelial Cells; Gene Expression Regulation, Enzymologic; Glutaminase; Glutamine; Heme Oxygenase-1; Humans; RNA Interference; Thiadiazoles; Veins
PubMed: 30144404
DOI: 10.1016/j.bcp.2018.08.032 -
Journal of Neuro-oncology Feb 2020Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection,...
PURPOSE
Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide. New approaches to treatment of glioblastoma, such as targeting metabolism, have been studied. The object of this study is to evaluate whether asparagine could be a new target for treatment of glioblastoma.
METHODS
We investigated a potential treatment for glioblastoma that targets the amino acid metabolism. U251, U87, and SF767 glioblastoma cells were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase hydrolyzes asparagine into aspartate and depletes asparagine. L-asparaginase has been used for the treatment of acute lymphoblastic leukemia. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase.
RESULTS
Cell viability was measured after 72 h of treatment. MTS assay showed that L-asparaginase suppressed the proliferation of U251, U87, and SF767 cells in a dose-dependent manner. DON also inhibited the proliferation of these cell lines in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. Exogenous asparagine rescued the effect of inhibition of proliferation by L-asparaginase and DON. The expression of asparagine synthetase mRNA was increased in cells treated with a combination of L-asparaginase and DON. This combined treatment also induced greater apoptosis and autophagy than did single-drug treatment.
CONCLUSION
The results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with glioblastoma.
Topics: Antineoplastic Agents; Asparaginase; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diazooxonorleucine; Glioblastoma; Humans
PubMed: 32020477
DOI: 10.1007/s11060-019-03351-4 -
British Journal of Anaesthesia Nov 2022Hypersensitivity to general anaesthetics predicts adverse postoperative outcomes in patients. Hypoxia exerts extensive pathophysiological effects on the brain; however,...
BACKGROUND
Hypersensitivity to general anaesthetics predicts adverse postoperative outcomes in patients. Hypoxia exerts extensive pathophysiological effects on the brain; however, whether hypoxia influences sevoflurane sensitivity and its underlying mechanisms remain poorly understood.
METHODS
Mice were acclimated to hypoxia (oxygen 10% for 8 h day) for 28 days and anaesthetised with sevoflurane; the effective concentrations for 50% of the animals (EC) showing loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR) were determined. Positron emission tomography-computed tomography, O-glycoproteomics, seahorse analysis, carbon-13 tracing, site-specific mutagenesis, and electrophysiological techniques were performed to explore the underlying mechanisms.
RESULTS
Compared with the control group, the hypoxia-acclimated mice required higher concentrations of sevoflurane to present LORR and LTWR (EC50: 1.61 [0.03]% vs 1.46 [0.04]%, P<0.01; EC50: 2.46 [0.14]% vs 2.22 [0.06]%, P<0.01). Hypoxia-induced reduction in sevoflurane sensitivity was correlated with elevation of protein O-linked N-acetylglucosamine (O-GlcNAc) modification in brain, especially in the thalamus, and could be abolished by 6-diazo-5-oxo-l-norleucine, a glutamine fructose-6-phosphate amidotransferase inhibitor, and mimicked by thiamet-G, a selective O-GlcNAcase inhibitor. Mechanistically, O-GlcNAcylation drives de novo synthesis of glutamine from glucose in astrocytes and promotes the glutamate-glutamine cycle, partially via glycolytic flux and activation of glutamine synthetase.
CONCLUSIONS
Intermittent hypoxia exposure decreased mouse sensitivity to sevoflurane anaesthesia through enhanced O-GlcNAc-dependent modulation of the glutamate-glutamine cycle in the brain.
Topics: Animals; Mice; Acetylglucosamine; Sevoflurane; Glutamine; Diazooxonorleucine; Glutamate-Ammonia Ligase; Brain; Hypoxia; Glucose; Anesthetics, General; Oxygen; Glutamates
PubMed: 36031420
DOI: 10.1016/j.bja.2022.06.041 -
Communications Biology 2019Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that...
Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management.
Topics: Animals; Body Weight; Brain; Brain Neoplasms; Caloric Restriction; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Diet, Ketogenic; Disease Models, Animal; Female; Fermentation; Glioblastoma; Glucose; Glutamine; Humans; Immunohistochemistry; Ketone Bodies; Ketones; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation
PubMed: 31149644
DOI: 10.1038/s42003-019-0455-x -
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 Genetics and Genomics = Yi... Sep 2011CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we...
CTP synthase is compartmentalized within a subcellular structure, termed the cytoophidium, in a range of organisms including bacteria, yeast, fruit fly and rat. Here we show that CTP synthase is also compartmentalized into cytoophidia in human cells. Surprisingly, the occurrence of cytoophidia in human cells increases upon treatment with a glutamine analog 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of glutamine-dependent enzymes including CTP synthase. Experiments in flies confirmed that DON globally promotes cytoophidium assembly. Clonal analysis via CTP synthase RNA interference in somatic cells indicates that CTP synthase expression level is critical for the formation of cytoophidia. Moreover, DON facilitates cytoophidium assembly even when CTP synthase level is low. A second glutamine analog azaserine also promotes cytoophidum formation. Our data demonstrate that glutamine analogs serve as useful tools in the study of cytoophidia.
Topics: Animals; Apoptosis; Azaserine; Carbon-Nitrogen Ligases; Cell Compartmentation; Cell Cycle; Diazooxonorleucine; Drosophila melanogaster; Enzyme Inhibitors; Female; Glutamine; HeLa Cells; Humans; Intracellular Space; Male; Oogenesis; RNA Interference
PubMed: 21930098
DOI: 10.1016/j.jgg.2011.08.004 -
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 -
Biochemical and Biophysical Research... Sep 1984Addition of NH4Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrogenase activity, which was recovered once the added NH+4...
Addition of NH4Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrogenase activity, which was recovered once the added NH+4 was exhausted from the medium. In the presence of inhibitors of ammonium assimilation, such as L-methionine-DL-sulfoximine, L-methionine sulfone or 6-diazo-5-oxo-L-norleucine, externally added NH+4 had no effect on nitrogenase activity and the newly-fixed nitrogen was excreted into the medium as NH+4. It is concluded that, in A. chroococcum, NH+4 must be assimilated to exert its short-term inhibitory effect on nitrogen fixation.
Topics: Ammonium Chloride; Azotobacter; Diazooxonorleucine; Methionine; Methionine Sulfoximine; Nitrogen Fixation; Nitrogenase
PubMed: 6593068
DOI: 10.1016/0006-291x(84)90248-1 -
Drug Discoveries & Therapeutics Nov 2023This work describes a novel artificial intelligence-based training and monitoring system (AITMS) that was used to control and prevent nosocomial infections (NIs) by...
This work describes a novel artificial intelligence-based training and monitoring system (AITMS) that was used to control and prevent nosocomial infections (NIs) by improving the skills of donning/removing personal protective equipment (PPE). The AITMS has two working modes, namely an AI-based protective equipment surveillance mode and an AI-based training mode, that were used for routine surveillance and training, respectively. Data revealed that the accuracy rate of donning/removing PPE improved as a result of the AITMS. Interestingly, the frequency of NIs decreased with the use of the AITMS. This study suggested the key role of using PPE in controlling and preventing NIs. Data preliminarily proved that appropriate donning/removing PPE may help to reduce the risk of NIs. In addition, the newest computerized technologies, such as AI, have proven to be useful in controlling and preventing NIs. These findings should helpful to formulate a better strategy against NIs in the future.
Topics: Humans; Artificial Intelligence; Pilot Projects; Cross Infection; Diazooxonorleucine; Hospitals
PubMed: 37673650
DOI: 10.5582/ddt.2023.01068 -
California Medicine Jul 1967These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California Medical Center, San Francisco. Taken from...
These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California Medical Center, San Francisco. Taken from transcriptions, they are prepared by Drs. Martin J. Cline and Hibbard E. Williams, Assistant Professors of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine.
Topics: Adenocarcinoma; Adrenal Gland Neoplasms; Animals; Breast Neoplasms; Diazooxonorleucine; Female; Humans; Hyperkalemia; Lung Neoplasms; Male; Rats
PubMed: 6068921
DOI: No ID Found