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American Journal of Physiology. Cell... Jun 2003Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or...
Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or presence of glutamine (0.5, 1.0, and 2.0 mM). Condensation of chromatin was reduced after 24 or 48 h of culture in the presence of glutamine compared with its absence as assessed by Hoechst 33342 staining. The level of Escherichia coli phagocytosis in the presence of glutamine was markedly increased compared with the level achieved by cells cultured in the absence of glutamine. Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine. Sensitive fluorochrome rhodamine 123, as determined by fluorescence-activated cell sorting and confocal microscopy, was used to monitor loss of the mitochondrial transmembrane potential. In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123. In the presence of 1.0 or 2.0 mM glutamine, the uptake of rhodamine was 20 or 38% higher, respectively. Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential. Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils.
Topics: Animals; Apoptosis; Cells, Cultured; Chromatin; Diazooxonorleucine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutamine; Humans; Male; Membrane Potentials; Mitochondria; Neutrophils; Phagocytosis; Phosphatidylserines; Rats; Rats, Wistar
PubMed: 12529242
DOI: 10.1152/ajpcell.00224.2002 -
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 -
American Journal of Physiology. Renal... Oct 2000Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by...
Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by glucose metabolism through the hexosamine biosynthesis pathway (HBP). The HBP converts fructose-6-phosphate to glucosamine-6-phosphate via the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA). We have investigated the effects of high glucose (HG, 25 mM) and glucosamine (GlcN, 1.5 mM) on the synthesis of the ECM protein laminin in a SV-40-transformed rat kidney mesangial (MES) cell line. The roles of protein kinases C (PKC) and A (PKA) in mediating laminin accumulation were also investigated. Treatment of MES cells with HG or GlcN for 48 h increased laminin levels in cellular extracts more than twofold compared with 5 mM glucose (low glucose; LG). The presence of the GFA inhibitor diazo-oxo-norleucine (DON, 10 microM) blocked HG but not GlcN-induced laminin synthesis. HG resulted in a time-dependent increase in total PKC and PKA activities, 57+/-11.3 (P < 0.01 vs. LG) and 85+/-17.4% (P < 0.01 vs. LG), respectively. GlcN had no effect on the total PKC activity; however, both glucose and glucosamine increased membrane-associated PKC activity by twofold compared with LG. GlcN stimulated total PKA activity by 47+/-8.4% (P < 0.01 vs. LG). Similarly, membrane- associated PKA activity was also increased by HG and GlcN approximately 1.8 and 1.5-fold, respectively. HG and GlcN increased cellular cAMP levels 2.2- and 3. 4-fold, respectively. Pharmacological downregulation of PKC by long-term incubation of MES cells with 0.5 microM phorbol 12-myristate 13-acetate (PMA) or inhibition of PKA activity by 2 microM H-8 blocked the effects of HG and GlcN on laminin synthesis. These results demonstrate that glucose-induced laminin synthesis in MES cells is mediated by flux through the HBP and that this stimulation involves PKC and PKA signaling pathways.
Topics: Animals; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Glomerular Mesangium; Glucosamine; Glucose; Hexosamines; Laminin; Protein Kinase C; Rats
PubMed: 10997914
DOI: 10.1152/ajprenal.2000.279.4.F646 -
The Biochemical Journal Jul 1999The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of...
The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.
Topics: Animals; Aspartic Acid; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Culture Media; Cytidine; Cytidine Triphosphate; DNA Damage; DNA-Binding Proteins; Diazooxonorleucine; Epithelial Cells; Gene Expression Regulation; Glutamine; Isoxazoles; Ketoglutaric Acids; Kidney Tubules, Proximal; RNA, Messenger; Salts; Swine; Transcription Factor CHOP; Transcription Factors; Uridine
PubMed: 10377266
DOI: No ID Found -
Journal of Bacteriology Jul 1997The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As...
The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.
Topics: Bacterial Proteins; Base Sequence; Biological Transport; Diazooxonorleucine; Glutamate-Ammonia Ligase; Methionine Sulfoximine; Molecular Sequence Data; Nitrogen; Nitrogen Fixation; Nucleotidyltransferases; PII Nitrogen Regulatory Proteins; Quaternary Ammonium Compounds; Rhodospirillum rubrum; Uridine
PubMed: 9209032
DOI: 10.1128/jb.179.13.4190-4194.1997 -
The Journal of Biological Chemistry Jun 1996Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of...
Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of glucose, and this effect of glucose can be mimicked by glucosamine. To determine whether the metabolism of glucose to glucosamine is required for this glucose effect, the rate-limiting step in glucose metabolism to glucosamine through the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) was blocked using pharmacological and antisense strategies. We found that blockage of GFAT activity or expression significantly blunted the glucose-induced increase of TGFalpha expression. Blockage of GFAT also resulted in a decreased RL2 signal on intracellular proteins as detected by Western blotting and indirect immunofluorescence. The RL2 monoclonal antibody recognizes an epitope on proteins that contain N-acetylglucosamine and thus is a measure of protein glycosylation. Conversely, treatment of the cells with glucose and glucosamine resulted in an increase in the RL2 epitope on intracellular proteins. These results indicate that the metabolism of glucose to glucosamine is necessary for the transcriptional stimulation of TGFalpha expression in vascular smooth muscle cells by glucose. Furthermore, the level of glycosylation of some intracellular proteins can be modulated in response to physiological changes in the extracellular glucose concentration and the net activity of GFAT.
Topics: Animals; Antibodies, Monoclonal; Aorta; Cells, Cultured; Diazooxonorleucine; Fluorescent Antibody Technique, Indirect; Glucosamine; Glucose; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycosylation; Kinetics; Luciferases; Male; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transcription, Genetic; Transforming Growth Factor alpha
PubMed: 8663078
DOI: 10.1074/jbc.271.25.15237 -
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 -
Annals of Surgery Dec 1993This overview on glutamine and cancer discusses the importance of glutamine for tumor growth, summarizes the alterations in interorgan glutamine metabolism that develop... (Review)
Review
OBJECTIVE
This overview on glutamine and cancer discusses the importance of glutamine for tumor growth, summarizes the alterations in interorgan glutamine metabolism that develop in the tumor-bearing host, and reviews the potential benefits of glutamine nutrition in the patient with cancer.
SUMMARY BACKGROUND DATA
Glutamine is the most abundant amino acid in the blood and tissues. It is essential for tumor growth and marked changes in organ glutamine metabolism are characteristic of the host with cancer. Because host glutamine depletion has adverse effects, it is important to study the regulation of glutamine metabolism in cancer and to evaluate the impact of glutamine nutrition in the tumor-bearing state.
METHODS
Data from a variety of investigations on glutamine metabolism and nutrition related to the host with cancer were compiled and summarized.
RESULTS
Numerous studies on glutamine metabolism in cancer indicate that many tumors are avid glutamine consumers in vivo and in vitro. As a consequence of progressive tumor growth, host glutamine depletion develops and becomes a hallmark. This glutamine depletion occurs in part because the tumor behaves as a "glutamine trap" but also because of cytokine-mediated alterations in glutamine metabolism in host tissues. Animal and human studies that have investigated the use of glutamine-supplemented nutrition in the host with cancer suggest that pharmacologic doses of dietary glutamine may be beneficial.
CONCLUSIONS
Understanding the control of glutamine metabolism in the tumor-bearing host not only improves the knowledge of metabolic regulation in the patient with cancer but also will lead to improved nutritional support regimens targeted to benefit the host.
Topics: Antimetabolites, Antineoplastic; Cell Division; Diazooxonorleucine; Glutamine; Humans; Intestinal Mucosa; Intestines; Isoxazoles; Lymphocytes; Muscles; Neoplasms; Parenteral Nutrition, Total; Randomized Controlled Trials as Topic
PubMed: 8257221
DOI: 10.1097/00000658-199312000-00004 -
Endocrine Journal Apr 1993We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase...
We have previously shown that methotrexate (MTX) and hydroxyurea (HU) stimulate expression of the human chorionic gonadotropin alpha and placental alkaline phosphatase genes and repress the expression of the c-myc oncogene in BeWo choriocarcinoma cells. In order to determine whether c-myc downregulation played a role in the induction of these placental genes, we treated BeWo choriocarcinoma cells with aphidicolin (APH), and 6-diazo-5-oxo-L-norleucine (DON), and compared the effects of these drugs with that of MTX. All of these drugs downregulate c-myc gene expression. At the doses used, both APH and DON repress c-myc expression to approximately the same extent, as well as inducing morphologic changes in BeWo similar to that of MTX, although neither stimulated hCG alpha expression. Both DON and APH stimulate placental alkaline phosphatase gene expression, but only MTX and DON stimulate cholesterol side chain cleavage enzyme gene expression. This indicates that the downregulation of c-myc gene expression is insufficient to stimulate the expression of all the placental genes stimulated by MTX.
Topics: Alkaline Phosphatase; Aphidicolin; Cholesterol; Choriocarcinoma; Chorionic Gonadotropin; DNA, Neoplasm; Diazooxonorleucine; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Methotrexate; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Neoplasms
PubMed: 7951513
DOI: 10.1507/endocrj.40.263 -
The Biochemical Journal Oct 1991Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves...
Glutamate synthase, a key enzyme in ammonia assimilation, has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The purification procedure involves ion-exchange chromatography, affinity chromatography and gel filtration. The recovery in the procedure is high (62%) and the specific activity is 21 mumol of NADPH oxidized/min per mg. The enzyme is specific for its substrates, and no activity was demonstrated with NADH or NH4+ ions substituting for NADPH and glutamine respectively. The enzyme is composed of two dissimilar subunits with molecular masses of 53 and 152 kDa, and it is shown that Cl- ions have an effect on the aggregation of the enzyme. Km values for the substrates are: NADPH, 16 microM; 2-oxoglutarate, 10 microM; and glutamine, 65 microM. The enzyme is inhibited by amidotransferase inhibitors at micromolar concentrations. The role of the enzyme in the metabolic regulation of nitrogenase is discussed.
Topics: Amino Acids; Azaserine; Chromatography, Liquid; Diazooxonorleucine; Electrophoresis, Polyacrylamide Gel; Glutamate Synthase; NAD; NADP; Nitrogen Fixation; Rhodospirillum rubrum; Substrate Specificity
PubMed: 1930133
DOI: 10.1042/bj2790151