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1.
Biotechnol Bioeng ; 119(7): 1712-1727, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35312045

RESUMO

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.


Assuntos
Glutamato-Amônia Ligase , Glutamina , Animais , Células CHO , Cricetinae , Cricetulus , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Glutamina/metabolismo , Imunoglobulina G/genética , Ácido Láctico/metabolismo , Metionina Sulfoximina/metabolismo , Metionina Sulfoximina/farmacologia
2.
Exp Eye Res ; 213: 108845, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34800480

RESUMO

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1ß and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.


Assuntos
Retinopatia Diabética/enzimologia , Inibidores Enzimáticos/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Interleucina-1beta/farmacologia , Retina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Retinopatia Diabética/patologia , Feminino , Glucose/farmacologia , Hiperglicemia/metabolismo , Ácido Iodoacético/farmacologia , L-Lactato Desidrogenase/metabolismo , Metionina Sulfoximina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Retina/enzimologia , Retina/patologia
3.
Int J Mol Sci ; 22(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34681786

RESUMO

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.


Assuntos
Encéfalo/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Metionina Sulfoximina/farmacologia , Convulsões , Animais , Encéfalo/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Lítio/efeitos adversos , Masculino , Metionina Sulfoximina/administração & dosagem , Pilocarpina/efeitos adversos , Ratos , Ratos Sprague-Dawley , Via Secretória/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Convulsões/patologia
4.
Oncol Rep ; 45(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33846803

RESUMO

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c­Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c­Myc protein expression, leading to a reduction in the levels of the canonical c­Myc isoform and an increase in the expression of c­Myc 1, a c­Myc isoform translated from an in­frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c­Myc expression as in glutamine­fed cells, with high levels of canonical c­Myc and c­Myc 1 almost undetectable. In asparagine­fed cells, global protein translation was higher than in glutamine­starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine­starved asparagine­fed cells, the inhibition of c­Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c­Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.


Assuntos
Asparagina/metabolismo , Transformação Celular Neoplásica/metabolismo , Glutamina/deficiência , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Inibidores de Cisteína Proteinase/farmacologia , Regulação Neoplásica da Expressão Gênica , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Humanos , Leupeptinas/farmacologia , Metionina Sulfoximina/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética
5.
Brain Res ; 1753: 147253, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33422530

RESUMO

The contribution of glutamatergic transmission to generation of initial convulsive seizures (CS) is debated. We tested whether pretreatment with a glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSO), affects the onset and progression of initial CS by cholinergic stimulus in juvenile rats. Male rats (24 days old, Sprague Dawley) sequentially received i.p. injections of lithium-carbonate, MSO, methyl-scopolamine, and pilocarpine (Pilo). Pilo was given 150 min after MSO. Animals were continuously monitored using the Racine scale, EEG/EMG and intrahippocampal glutamate (Glu) biosensors. GS activity as measured in hippocampal homogenates, was not altered by MSO at 150 min, showed initial, varied inhibition at 165 (15 min post-Pilo), and dropped down to 11% of control at 60 min post-Pilo, whereas GS protein expression remained unaltered throughout. Pilo did neither modulate the effect of MSO on GS activity nor affect GS activity itself, at any time point. MSO reduced from 32% to 4% the number of animals showing CS during the first 12 min post-Pilo, delayed by ~6 min the appearance of electrographic seizures, and tended to decrease EMG power during ~15 min post-Pilo. The results indicate that MSO impairs an aspect of glutamatergic transmission involved in the transition from the first cholinergic stimulus to the onset of seizures. A continuous rise of extracellular Glu lasting 60 min was insignificantly affected by MSO, leaving the nature of the Glu pool(s) involved in altered glutamatergic transmission undefined.


Assuntos
Encéfalo/efeitos dos fármacos , Glutamato-Amônia Ligase/efeitos dos fármacos , Pilocarpina/farmacologia , Convulsões , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glutamina/metabolismo , Masculino , Metionina Sulfoximina/farmacologia , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico
6.
J Biotechnol ; 325: 389-394, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32961202

RESUMO

Bioavailable glutamine (Gln) is critical for metabolism, intestinal health, immune function, and cell signaling. Routine measurement of serum Gln concentrations could facilitate improved diagnosis and treatment of severe infections, anorexia nervosa, chronic kidney disease, diabetes, and cancer. Current methods for quantifying tissue Gln concentrations rely mainly on HPLC, which requires extensive sample preparation and expensive equipment. Consequently, patient Gln levels may be clinically underutilized. Cell-free protein synthesis (CFPS) is an emerging sensing platform with promising clinical applications, including detection of hormones, amino acids, nucleic acids, and other biomarkers. In this work, in vitro E. coli amino acid metabolism is engineered with methionine sulfoximine to inhibit glutamine synthetase and create a CFPS Gln sensor. The sensor features a strong signal-to-noise ratio and a detection range ideally suited to physiological Gln concentrations. Furthermore, it quantifies Gln concentration in the presence of human serum. This work demonstrates that CFPS reactions which harness the metabolic power of E. coli lysate may be engineered to detect clinically relevant analytes in human samples. This approach could lead to transformative point-of-care diagnostics and improved treatment regimens for a variety of diseases including cancer, diabetes, anorexia nervosa, chronic kidney disease, and severe infections.


Assuntos
Escherichia coli , Glutamina , Aminoácidos , Escherichia coli/genética , Glutamato-Amônia Ligase , Humanos , Metionina Sulfoximina
7.
Fish Shellfish Immunol ; 101: 198-204, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32251762

RESUMO

Ammonia is toxic to most bony fishes. However, little information is available on the toxicology mechanisms induced by ammonia and the means to mitigate the effects by various fishes. In this study, four groups of experiments were designed and carried out to test the response of dolly varden char to ammonia toxicity and their mitigation through methionine sulfoximine (MSO). NaCl group was injected with NaCl, NH3 group was injected with ammonium acetate, NH3+MSO group was injected with ammonium acetate and MSO, MSO group was injected with MSO. Results showed that ammonia toxicity could lead to blood deterioration (elevation in white blood cell and blood ammonia), free amino acid imbalance (elevation in glutamine, glutamate, arginine and ornithine, coupled with reduction of citrulline and aspartate), ammonia metabolism enzyme activity inhibition (reduction in carbamyl phosphate synthetase, ornithine transcarbamylase and arginase), oxidative stress (reduction in superoxide dismutase, catalase and glutathione peroxidase) and immunosuppression (reduction in lysozyme, 50% hemolytic complement, total immunoglobulin and phagocytic index), but the MSO can eliminate fatal effect of oxidative damage. In addition, ammonia poisoning could induce down-regulation of antioxidant enzymes coding genes (SOD, CAT and GPx) and up-regulation of inflammatory cytokine genes (TNFα, IL-1ß and IL-8) transcription, suggesting that immunosuppression and inflammation may relate to oxidative stress in fish.


Assuntos
Aminoácidos/metabolismo , Amônia/envenenamento , Expressão Gênica/imunologia , Imunidade , Metionina Sulfoximina/administração & dosagem , Substâncias Protetoras/administração & dosagem , Truta/imunologia , Animais , Análise Química do Sangue/veterinária , Truta/sangue , Truta/genética
8.
Chemistry ; 26(19): 4378-4388, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961028

RESUMO

A short synthetic approach with broad scope to access five- to seven-membered cyclic sulfoximines in only two to three steps from readily available thiophenols is reported. Thus, simple building blocks were converted to complex molecular structures by a sequence of S-alkylation and one-pot sulfoximine formation, followed by intramolecular cyclization. Seventeen structurally diverse cyclic sulfoximines were prepared in high overall yields. In vitro evaluation of these underrepresented, three-dimensional, cyclic sulfoximines with respect to properties relevant to medicinal chemistry did not reveal any intrinsic flaw for application in drug discovery.


Assuntos
Descoberta de Drogas/métodos , Metionina Sulfoximina/síntese química , Alquilação , Química Farmacêutica , Ciclização , Metionina Sulfoximina/química , Estrutura Molecular
9.
BMC Plant Biol ; 19(1): 425, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615403

RESUMO

BACKGROUND: Nitrogen (N) nutrition significantly affected metabolism and accumulation of quality-related compounds in tea plant (Camellia sinensis L.). Little is known about the physiological and molecular mechanisms underlying the effects of short-term repression of N metabolism on tea roots and leaves for a short time. RESULTS: In this study, we subjected tea plants to a specific inhibitor of glutamine synthetase (GS), methionine sulfoximine (MSX), for a short time (30 min) and investigated the effect of the inhibition of N metabolism on the transcriptome and metabolome of quality-related compounds. Our results showed that GS activities in tea roots and leaves were significantly inhibited upon MSX treatment, and both tissue types showed a sensitive metabolic response to GS inhibition. In tea leaves, the hydrolysis of theanine decreased with the increase in theanine and free ammonium content. The biosynthesis of all other amino acids was repressed, and the content of N-containing lipids declined, suggesting that short-term inhibition of GS reduces the level of N reutilization in tea leaves. Metabolites related to glycolysis and the tricarboxylic acid (TCA) cycle accumulated after GS repression, whereas the content of amino acids such as glycine, serine, isoleucine, threonine, leucine, and valine declined in the MXS treated group. We speculate that the biosynthesis of amino acids is affected by glycolysis and the TCA cycle in a feedback loop. CONCLUSIONS: Overall, our data suggest that GS repression in tea plant leads to the reprogramming of amino acid and lipid metabolic pathways.


Assuntos
Aminoácidos/metabolismo , Camellia sinensis/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Metabolismo dos Lipídeos , Metionina Sulfoximina/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Camellia sinensis/efeitos dos fármacos , Camellia sinensis/enzimologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo
10.
J Biol Chem ; 294(48): 18244-18255, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31641022

RESUMO

The evolutionarily conserved TOR complex 1 (TORC1) activates cell growth and proliferation in response to nutritional signals. In the fission yeast Schizosaccharomyces pombe, TORC1 is essential for vegetative growth, and its activity is regulated in response to nitrogen quantity and quality. Yet, how TORC1 senses nitrogen is poorly understood. Rapamycin, a specific TOR inhibitor, inhibits growth in S. pombe only under conditions in which the activity of TORC1 is compromised. In a genetic screen for rapamycin-sensitive mutations, we isolated caa1-1, a loss-of-function mutation of the cytosolic form of aspartate aminotransferase (Caa1). We demonstrate that loss of caa1 + partially mimics loss of TORC1 activity and that Caa1 is required for full TORC1 activity. Disruption of caa1 + resulted in aspartate auxotrophy, a finding that prompted us to assess the role of aspartate in TORC1 activation. We found that the amino acids glutamine, asparagine, arginine, aspartate, and serine activate TORC1 most efficiently following nitrogen starvation. The glutamine synthetase inhibitor l-methionine sulfoximine abolished the ability of asparagine, arginine, aspartate, or serine, but not that of glutamine, to induce TORC1 activity, consistent with a central role for glutamine in activating TORC1. Neither addition of aspartate nor addition of glutamine restored TORC1 activity in caa1-deleted cells or in cells carrying a Caa1 variant with a catalytic site substitution, suggesting that the catalytic activity of Caa1 is required for TORC1 activation. Taken together, our results reveal the contribution of the key metabolic enzyme Caa1 to TORC1 activity in S. pombe.


Assuntos
Aspartato Aminotransferases/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Arginina/farmacologia , Asparagina/farmacologia , Aspartato Aminotransferases/metabolismo , Ácido Aspártico/farmacologia , Citosol/enzimologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina Sulfoximina/farmacologia , Nitrogênio/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sirolimo/farmacologia
11.
Biotechnol Prog ; 35(5): e2856, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31148368

RESUMO

Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate-without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions.


Assuntos
Técnicas de Cultura de Células/métodos , Glutamato-Amônia Ligase , Glutamina/metabolismo , Serina/metabolismo , Animais , Células CHO , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Cricetinae , Cricetulus , Meios de Cultura/química , Meios de Cultura/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Glutamina/análise , Metionina Sulfoximina
12.
MAbs ; 11(5): 965-976, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043114

RESUMO

Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's primary means of manufacturing therapeutic proteins, including monoclonal antibodies. The major challenge in cell line development for the production of recombinant biopharmaceuticals lies in generating and isolating rare high-producing stable clones, amongst thousands of low-producing or unstable clones, in a short period of time. One approach to accomplish this is to use the glutamine synthetase (GS) selection system, together with the GS inhibitor, methionine sulfoximine (MSX). However, MSX can only increase protein productivity to a limited extent. Often productivity will drop when MSX is removed from the system. We evaluated a congenital GS mutation, R324C, which causes glutamine deficiency in human as an attenuated selection marker for CHO cell line generation. We also created a panel of GS mutants with diminished GS activity. Our results demonstrated that using attenuated GS mutants as selection markers significantly increased antibody production of stably transfected pools. Furthermore, these stably transfected pools sustained high productivity levels for an extended period of time, whereas cells transfected with wild-type GS lost considerable protein productivity over time, particularly after MSX was removed. In summary, the use of attenuated GS as a selection marker in CHO cell line development bypasses the need for MSX, and generates stable clones with significantly higher antibody productivity.Abbreviations: CHO: Chinese hamster ovary; CMV: Cytomegalovirus; DHFR: Dihydrofolate reductase; GFP: Green fluorescent protein; GOI: gene-of-interest; GS: Glutamine synthetase; IRES: internal ribosomal entry site; MSX: Methionine sulfoximine; MTX: Methotrexate; psGS: pseudoGS; RVDs: Repeated variable di-residues; TALENs: transcription activator-like effector nucleases; VCD: Viable cell density; ZFNs: zinc finger nucleases.


Assuntos
Anticorpos Monoclonais/biossíntese , Células CHO/imunologia , Glutamato-Amônia Ligase/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Células CHO/enzimologia , Cricetulus , Humanos , Metionina Sulfoximina/farmacologia , Transfecção
13.
Plant Signal Behav ; 14(4): e1582263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30810449

RESUMO

Foliar uptake of nitrogen dioxide (NO2) is governed by its reactive absorption mechanism, by which NO2 molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO2 remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO2, as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm 15N-labeled NO2 for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO2 using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO2 in leaves without MSX treatment was 587.0 nmol NO2/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO2 by half. Thus, half of the foliar uptake of NO2 is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO2 is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.


Assuntos
Arabidopsis/metabolismo , Ciclo do Nitrogênio/fisiologia , Dióxido de Nitrogênio/metabolismo , Amônia/metabolismo , Ácido Ascórbico/metabolismo , Metionina Sulfoximina/farmacologia , Nitritos/metabolismo , Nitrogênio/metabolismo , Ácido Nitroso/metabolismo , Folhas de Planta/metabolismo
14.
Sci Rep ; 9(1): 252, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670758

RESUMO

Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.


Assuntos
Glutamato-Amônia Ligase/metabolismo , Neurogênese/fisiologia , Memória Espacial/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Região CA3 Hipocampal/crescimento & desenvolvimento , Região CA3 Hipocampal/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Masculino , Metionina Sulfoximina/farmacologia , Camundongos , Modelos Animais , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Memória Espacial/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
15.
Sci Rep ; 8(1): 5361, 2018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29599455

RESUMO

To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 µM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 µM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 µM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.


Assuntos
Anticorpos Monoclonais/biossíntese , Células Clonais/metabolismo , Glutamato-Amônia Ligase , Metionina Sulfoximina/metabolismo , Amônia/metabolismo , Animais , Células CHO , Cricetulus , Técnicas de Inativação de Genes , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Ácido Láctico/metabolismo , Metionina Sulfoximina/química
16.
Mol Pain ; 14: 1744806918763270, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29448913

RESUMO

Background The mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle hyperalgesia remain largely underinvestigated. In the present study, we aimed to determine whether masseter muscle contraction induced by daily electrical stimulation influences the mechanical head-withdrawal threshold and genioglossus electromyography activity caused by the application of capsaicin to the upper first molar tooth pulp. We further investigated whether astroglial glutamine synthesis is involved in first molar tooth pulp hypersensitivity associated with masseter muscle contraction. Methods The first molar tooth pulp was treated with capsaicin or vehicle in masseter muscle contraction or sham rats, following which the astroglial glutamine synthetase inhibitor methionine sulfoximine or Phosphate buffered saline (PBS) was applied. Astroglial activation was assessed via immunohistochemistry. Results The mechanical head-withdrawal threshold of the ipsilateral masseter muscle was significantly decreased in masseter muscle contraction rats than in sham rats. Genioglossus electromyography activity was significantly higher in masseter muscle contraction rats than sham rats. Glial fibrillary acidic protein-immunoreactive cell density was significantly higher in masseter muscle contraction rats than in sham rats. Administration of methionine sulfoximine induced no significant changes in the density of glial fibrillary acidic protein-immunoreactive cells relative to PBS treatment. However, mechanical head-withdrawal threshold was significantly higher in masseter muscle contraction rats than PBS-treated rats after methionine sulfoximine administration. Genioglossus electromyography activity following first molar tooth pulp capsaicin treatment was significantly lower in methionine sulfoximine-treated rats than in PBS-treated rats. In the ipsilateral region, the total number of phosphorylated extracellular signal-regulated protein kinase immunoreactive cells in the medullary dorsal horn was significantly smaller upon first molar tooth pulp capsaicin application in methionine sulfoximine-treated rats than in PBS-treated rats. Conclusions Our results suggest that masseter muscle contraction induces astroglial activation, and that this activation spreads from caudal to the obex in the medullary dorsal horn, resulting in enhanced neuronal excitability associated with astroglial glutamine synthesis in medullary dorsal horn neurons receiving inputs from the tooth pulp. These findings provide significant insight into the mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle contraction.


Assuntos
Astrócitos/metabolismo , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Glutamina/metabolismo , Músculo Masseter/fisiopatologia , Bulbo/metabolismo , Contração Muscular , Animais , Astrócitos/efeitos dos fármacos , Capsaicina/farmacologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/fisiopatologia , Estimulação Elétrica , Eletromiografia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Masculino , Músculo Masseter/efeitos dos fármacos , Músculo Masseter/patologia , Bulbo/efeitos dos fármacos , Bulbo/fisiopatologia , Metionina Sulfoximina/administração & dosagem , Metionina Sulfoximina/farmacologia , Dente Molar/patologia , Contração Muscular/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/metabolismo , Ratos Sprague-Dawley
17.
Biotechnol Prog ; 34(2): 463-477, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29314708

RESUMO

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.


Assuntos
Anticorpos Monoclonais/biossíntese , Células CHO/citologia , Criopreservação , Glutamato-Amônia Ligase/química , Animais , Anticorpos Monoclonais/química , Cricetulus , Citometria de Fluxo , Glutamato-Amônia Ligase/genética , Metionina Sulfoximina/química
18.
Artigo em Inglês | MEDLINE | ID: mdl-29359358

RESUMO

In insects, glutamine synthetase (GS), a key enzyme in the synthesis of glutamine, has been reported to be associated with embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on postembryonic development. In this study, we demonstrate that blocking the activity of GS in the oriental fruit fly (Bactrocera dorsalis) with use of a GS-specific inhibitor (L-methionine S-sulfoximine), led to a significant delay in larval development, pupal weight loss, and inhibition of pupation. We further identify cloned and characterized two GS genes (BdGS-c and BdGS-m) from B. dorsalis. The two GS genes identified in B. dorsalis were predicted to be located in the cytosol (BdGS-c) and mitochondria (BdGS-m), and homology analysis indicated that both genes were similar to homologs from other Dipterans, such as Drosophila melanogaster and Aedes aegypti. BdGS-c was highly expressed in the larval stages, suggesting that cytosolic GS plays a predominant role in larval development. Furthermore, RNA interference experiments against BdGS-c, to specifically decrease the expression of cytosolic GS, resulted in delay in larval development as well as pupal weight loss. This study presents the prominent role played by BdGS-c in regulating larval development and suggests that the observed effect could have been modulated through ecdysteroid synthesis, agreeing with the reduced expression of the halloween gene spook. Also, the direct effects of BdGS-c silencing on B. dorsalis, such as larval lethality, delayed pupation, and late emergence, can be further exploited as novel insecticide target in the context of pest management.


Assuntos
Glutamato-Amônia Ligase/metabolismo , Tephritidae/enzimologia , Tephritidae/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Feminino , Glutamato-Amônia Ligase/genética , Proteínas de Insetos/metabolismo , Larva/enzimologia , Larva/crescimento & desenvolvimento , Metionina Sulfoximina , Filogenia , Interferência de RNA , Tephritidae/genética
19.
Biotechnol J ; 13(1)2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28745430

RESUMO

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression.


Assuntos
Anticorpos Monoclonais/biossíntese , DNA/genética , Glutamato-Amônia Ligase/genética , Nucleosídeos/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Regulação da Expressão Gênica/genética , Glutamato-Amônia Ligase/antagonistas & inibidores , Metionina Sulfoximina/farmacologia , Metotrexato/farmacologia , Mutagênese/genética , Transfecção
20.
J Plant Physiol ; 218: 167-170, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28866325

RESUMO

Pseudomonas syringae pv. tabaci causes wildfire disease by the action of tabtoxinine-ß-lactam (TßL), a non-specific bacterial toxin. To better understand the molecular mechanisms of wildfire disease and its development, we focused on the phosphoinositide 3-kinase in Nicotiana benthamiana (NbPI3K) and its potential role in the disease outbreak, using l-methionine sulfoximine (MSX) as an easily accessible mimic of the TßL action. The NbPI3K-silenced plants showed accelerated induction of cell death and necrotic lesion formation by MSX, and the expression of hin1, marker gene for the programmed cell death, was strongly induced in the plants. However, the accumulation of ammonium ions, caused by MSX inhibition of glutamine sythetase activity, was not affected by the NbPI3K-silencing. Interestingly, the expression of PR-1a, a marker gene for salicylic acid (SA) innate immunity signaling, and accumulation of SA were both enhanced in the NbPI3K-silenced plants. Accordingly, the acceleration of MSX-induced cell death by NbPI3K-silencing was reduced in NahG plants, and by double silencing of NbPI3K together with the NbICS1 encoding a SA-biosynthetic enzyme. As silencing of NbPI3K accelerated the TßL-induced necrotic lesions, and lesions of wildfire disease caused by P. syringae pv. tabaci, these results suggest that the NbPI3K-related pathway might act as a negative regulator of cell death during development of wildfire disease that involves SA-dependent signaling pathway downstream of TßL action in N. benthamiana.


Assuntos
Morte Celular , Metionina Sulfoximina/metabolismo , Fosfatidilinositol 3-Quinase/genética , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Transdução de Sinais , Tabaco/fisiologia , Inativação Gênica , Fosfatidilinositol 3-Quinase/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Pseudomonas syringae/fisiologia , Tabaco/genética
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