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Journal of Bacteriology Sep 1987The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense...
The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.
Topics: Alanine; Ammonia; Glutamate Dehydrogenase; Glutamate Synthase; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Gram-Negative Bacteria; Nitrogen; Transaminases
PubMed: 2887545
DOI: 10.1128/jb.169.9.4211-4214.1987 -
Clinical Cancer Research : An Official... Oct 2019Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular...
PURPOSE
Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT.
EXPERIMENTAL DESIGN
Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT.
RESULTS
Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT.
CONCLUSIONS
Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Diazooxonorleucine; Female; Glutamine; Humans; Metabolome; Mice; Mice, Nude; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rhabdoid Tumor; Teratoma; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31300448
DOI: 10.1158/1078-0432.CCR-19-0189 -
Proceedings of the National Academy of... Oct 2015The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective...
The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.
Topics: Animals; Antimalarials; Blood-Brain Barrier; Diazooxonorleucine; Glutamine; Malaria, Cerebral; Malaria, Falciparum; Mice
PubMed: 26438846
DOI: 10.1073/pnas.1516544112 -
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 -
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 -
Journal of Neurovirology Apr 2015Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits....
Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.
Topics: Alphavirus Infections; Animals; Antimetabolites, Antineoplastic; Behavior, Animal; Diazooxonorleucine; Disease Models, Animal; Encephalitis, Viral; Enzyme-Linked Immunosorbent Assay; Glutamine; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Sindbis Virus
PubMed: 25645378
DOI: 10.1007/s13365-015-0314-6 -
Proceedings of the National Academy of... May 2001The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low...
The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.
Topics: Adenosine Triphosphate; Animals; Carbon-Nitrogen Ligases; Cells, Cultured; Cytidine; Cytidine Triphosphate; Diazooxonorleucine; Enzyme Inhibitors; Fibroblasts; Guanine; Guanosine Triphosphate; Humans; Hypoxanthines; Intracellular Fluid; Isoxazoles; Mice; Mice, Inbred BALB C; Trypanocidal Agents; Trypanosoma brucei brucei; Trypanosomiasis, African; Uridine Triphosphate
PubMed: 11353848
DOI: 10.1073/pnas.111139498 -
Journal of Virology Jul 1994The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A...
The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed.
Topics: Base Sequence; Cell Membrane; DNA Primers; Diazooxonorleucine; Fluorescent Antibody Technique; Glycosylation; HeLa Cells; Humans; Molecular Sequence Data; Poliovirus; Protein Biosynthesis; Protein Precursors; RNA, Viral; Recombinant Proteins; Subcellular Fractions; Viral Core Proteins
PubMed: 8207820
DOI: 10.1128/JVI.68.7.4468-4477.1994 -
Neoplasia (New York, N.Y.) Jan 2010Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease...
Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease is significantly more prevalent and severe in postmenopausal women. We postulated that menopause could augment ovarian cancer progression through the effects of gonadotropins on multifocal seeding to the mesothelial layer lining the peritoneum. This seeding is mediated by integrins as well as by CD44 interaction with hyaluronan (HA). Here, we report the effect of gonadotropins on HA synthesis and degradation and on peritoneal adhesion. A significant concentration- and time-dependent induction in expression levels of HA synthases (HASs) and hyaluronidases (Hyals) was observed in vitro on stimulation of human epithelial ovarian carcinoma cells by gonadotropins. Hormonal regulation of HA-mediated adhesion was manifested in vivo as well, by fluorescence microscopy of stained MLS multicellular tumor spheroids. The number of spheroids adhered to the mesothelium of ovariectomized CD-1 nude mice 9.5 hours after intraperitoneal insertion was significantly higher than in nonovariectomized mice. Inhibition of HA synthesis by 6-diazo-5-oxo-1-norleucine (DON) both in spheroids and ovariectomized mice significantly reduced the number of adhered spheroids. Thus, the change in the hormonal environment during menopause assists in HA-dependent adherence of ovarian cancer spheroids onto the peritoneum. However, HA is antiangiogenic and it can significantly suppress tumor progression. Accordingly, angiogenesis of the adhered spheroids was significantly elevated in DON-treated tumors. These results can explain the selective pressure that can lead to simultaneously increased tumor expression of both HASs and Hyals.
Topics: Animals; Antimetabolites, Antineoplastic; Cell Adhesion; Cell Line, Tumor; Diazooxonorleucine; Dose-Response Relationship, Drug; Female; Follicle Stimulating Hormone; Glucuronosyltransferase; Gonadotropins; Humans; Hyaluronan Synthases; Hyaluronic Acid; Hyaluronoglucosaminidase; In Situ Hybridization; Luteinizing Hormone; Mice; Mice, Nude; Microscopy, Fluorescence; Neovascularization, Pathologic; Ovarian Neoplasms; Ovariectomy; Peritoneum; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Xenograft Model Antitumor Assays
PubMed: 20072653
DOI: 10.1593/neo.91272 -
Molecular Carcinogenesis Jun 2019Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy....
Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.
Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Diazooxonorleucine; Down-Regulation; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Heat-Shock Proteins; Hexosamines; Humans; Lung Neoplasms
PubMed: 30790354
DOI: 10.1002/mc.22992