scholarly journals A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats

1990 ◽  
Vol 270 (2) ◽  
pp. 473-481 ◽  
Author(s):  
N A Farrow ◽  
K Kanamori ◽  
B D Ross ◽  
F Parivar
Keyword(s):  
Nitrogen Atom ◽  
Glutamine Synthetase ◽  
De Novo ◽  
Specific Activity ◽  
Alternative Pathway ◽  
Fold Increase ◽  
Ammonia Removal ◽  
Heterogeneous Distribution ◽  
Ammonia Detoxification

1. Rats were infused with 15NH4+ or L-[15N]alanine to induce hyperammonaemia, a potential cause of hepatic encephalopathy. HClO4 extracts of freeze-clamped brain, liver and kidney were analysed by 15N-n.m.r. spectroscopy in combination with biochemical assays to investigate the effects of hyperammonaemia on tissue concentrations of ammonia, glutamine, glutamate and urea. 2. 15NH4+ infusion resulted in a 36-fold increase in the concentration of blood ammonia. Cerebral glutamine concentration increased, with 15NH4+ incorporated predominantly into the gamma-nitrogen atom of glutamine. Incorporation into glutamate was very low. Cerebral ammonia concentration increased 5-10-fold. The results suggest that the capacity of glutamine synthetase for ammonia detoxification was saturated. 3. Pretreatment with the glutamine synthetase inhibitor L-methionine DL-sulphoximine resulted in 84% inhibition of [gamma-15N]glutamine synthesis, but incorporation of 15N into other metabolites was not observed. The result suggests that no major alternative pathway for ammonia detoxification, other than glutamine synthetase, exists in rat brain. 4. In the liver 15NH4+ was incorporated into urea, glutamine, glutamate and alanine. The specific activity of 15N was higher in the gamma-nitrogen atom of glutamine than in urea. A similar pattern was observed when [15N]alanine was infused. The results are discussed in terms of the near-equilibrium states of the reactions involved in glutamate and alanine formation, heterogeneous distribution in the liver lobules of the enzymes involved in ammonia removal and their different affinities for ammonia. 5. Synthesis of glutamine, glutamate and hippurate de novo was observed in kidney. Hippurate, as well as 15NH4+, was contributed by co-extracted urine. 6. The potential utility and limitations of 15N n.m.r. for studies of mammalian metabolism in vivo are discussed.

scholarly journals De Novo Design of Granulopoietic Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Julia Skokowa ◽  
Mohammad Elgamacy ◽  
Patrick Müller
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Structural Information ◽  
Coiled Coil ◽  
Design Strategies ◽  
Nmr Structure Determination ◽  
Binding Epitope

Protein therapeutics are clinically developed and used as minorly engineered forms of their natural templates. This direct adoption of natural proteins in therapeutic contexts very frequently faces major challenges, including instability, poor solubility, and aggregation, which may result in undesired clinical outcomes. In contrast to classical protein engineering techniques, de novo protein design enables the introduction of radical sequence and structure manipulations, which can be used to address these challenges. In this work, we test the utility of two different design strategies to design novel granulopoietic proteins, using structural information from human granulocyte-colony stimulating factor (hG-CSF) as a template. The two strategies are: (1) An epitope rescaffolding where we migrate a tertiary structural epitope to simpler, idealised, proteins scaffolds (Fig. 1A-C), and (2) a topological refactoring strategy, where we change the protein fold by rearranging connections across the secondary structures and optimised the designed sequence of the new fold (Fig. 1A,D,E). Testing only eight designs, we obtained novel granulopoietic proteins that bind to the G-CSF receptor, have nanomolar activity in cell-based assays, and were highly thermostable and protease-resistant. NMR structure determination showed three designs to match their designed coordinates within less than 2.5 Å. While the designs possessed starkly different sequence and structure from the native G-CSF, they showed very specific activity in differentiating primary human haematopoietic stem cells into fully mature granulocytes. Morever, one design shows significant and specific activity in vivo in zebrafish and mice. These results are prospectively directing us to investigate the role of dimerisation geometry of G-GCSF receptor on activation magnitude and downstream signalling pathways. More broadly, the results also motivate our ongoing work on to design other heamatopoietic agents. In conclusion, our findings highlight the utility of computational protein design as a highly effective and guided means for discovering nover receptor modulators, and to obtain new mechanistic information about the target molecule. Figure 1. Two different strategies to generate superfolding G-CSF designs. (A) X-ray structure of G-CSF (orange) bound to its cognate receptor (red) through its binding epitope (blue). According to the epitope rescaffolding strategy, (B) the critical binding epitope residues were disembodied and used as a geometric search query against the entire Protein Data Bank (PDB) to retrieve structurally compatible scaffolds. The top six compatible scaffolds structures are shown in cartoon representation. (C) The top two templates chosen for sequence design, were a de novo designed coiled-coil and a four-helix bundle with unknown function. The binding epitopes were grafted, and the scaffolds were optimised to rigidly host the guest epitope. (D-E) According to the topological refactoring strategy (D) the topology of the native G-CSF was rewired from around the fixed binding epitope, and then was further mutated to idealise the core residues (blue volume (E)) and residues distal from the binding epitope (orange crust (E)). Both strategies aimed at simplifying the topology, reducing the size, and rigidifying the bound epitope conformation through alternate means. Figure 1 Disclosures No relevant conflicts of interest to declare.

Encapsulation of glutamine synthetase in mouse erythrocytes: a new procedure for ammonia detoxification

2008 ◽  
Vol 86 (6) ◽  
pp. 469-476 ◽  
Author(s):  
Elena A. Kosenko ◽  
Natalia I. Venediktova ◽  
Andrey A. Kudryavtsev ◽  
Fazoil I. Ataullakhanov ◽  
Yury G. Kaminsky ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Glutamine Synthetase ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Cell Recovery ◽  
Ammonia Detoxification ◽  
Neurological Alterations

There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 ± 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations.

scholarly journals The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

1974 ◽  
Vol 137 (3) ◽  
pp. 567-574 ◽  
Author(s):  
A. B. Graham ◽  
B. G. Woodcock ◽  
G. C. Wood
Keyword(s):  
Specific Activity ◽  
Phospholipid Composition ◽  
Fold Increase ◽  
Protein Deficiency ◽  
Male Rats ◽  
Striking Difference ◽  
Uridine Diphosphate ◽  
Microsomal Membranes ◽  
Microsomal Phospholipid

After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.

scholarly journals Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments

1991 ◽  
Vol 277 (3) ◽  
pp. 863-868 ◽  
Author(s):  
D Sömjen ◽  
K D Schlüter ◽  
E Wingender ◽  
H Mayer ◽  
A M Kaye
Keyword(s):  
Parathyroid Hormone ◽  
Bone Resorption ◽  
Thymidine Incorporation ◽  
Specific Activity ◽  
Fold Increase ◽  
Dependent Manner ◽  
Equimolar Concentration ◽  
Dose Dependent

We have found, in previous studies in vitro using skeletal derived cell cultures, that mid-region fragments of human parathyroid hormone (hPTH) stimulate [3H]thymidine incorporation into DNA and increase the specific activity of the brain-type isoenzyme of creatine kinase (CK). These changes occurred without an increase in cyclic AMP formation which is linked to bone resorption. In this study, we found that the mid-region fragment hPTH-(28-48) stimulated CK activity in diaphysis, epiphysis and kidney in a time- and dose-dependent manner, parallel to the effects of the whole molecule bovine (b)PTH-(1-84) and the fully active fragment hPTH-(1-34). The increase caused by hPTH-(28-48) at a dose of 1.25 micrograms/rat was not less than the 2-fold increase caused by a roughly equimolar concentration bPTH-(1-84). A significant increase was reached at 1 h after intraperitoneal injection in all cases. All three sequences of PTH caused an increase in [3H]thymidine incorporation into DNA in diaphysis and epiphysis, but not in kidney, 24 h after injection. A fragment further towards the C-terminal, hPTH-(34-47), was inactive compared with an equimolar concentration of the fragment hPTH-(25-39), which stimulated both CK activity and DNA synthesis. These results in vivo are in line with previous findings in vitro; they provide further support for the suggestion that mid-region fragments of the PTH molecule could be used to induce bone formation without incurring the deleterious effect of bone resorption.

scholarly journals Methotrexate Increases Skeletal Muscle GLUT4 Expression and Improves Metabolic Control in Experimental Diabetes

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Giuseppina T. Russo ◽  
Letteria Minutoli ◽  
Alessandra Bitto ◽  
Domenica Altavilla ◽  
Eugenio Alessi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Metabolic Control ◽  
Glucose Transporter ◽  
De Novo ◽  
Fold Increase ◽  
Low Doses ◽  
Glucose Levels ◽  
Glut4 Expression ◽  
Experimental Type

Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purinede novosynthesis, and its tissue concentrations can be increased,in vivo, by low doses of methotrexate (MTX) through the inhibition of the enzyme AICAR transformylase. We report here the first evidence that, in experimental type 2 diabetes, chronic treatment with low doses of MTX increases skeletal muscle GLUT4 expression and improves metabolic control. MTX (0.5 mg/kg body weight) or vehicle was administered intraperitoneally, once a week for 4 weeks, to genetically diabetic female C57BL/KsJ-m+/+Leptdbmice (db+/db+) and their normoglycemic littermates (db+/+m). In thedb+/db+mice, MTX treatment was associated with a ∼2-fold increase in skeletal muscle GLUT4 protein concentration and a >4-fold increase in GLUT4 mRNA expression (P<0.01, all), as compared to vehicle-treated mice; no significant differences were noted in controls. MTX treatment was also associated with a significant reduction of glucose and insulin serum concentrations in diabetic mice (P<0.001), and glucose levels only (P<0.05) in controls. These data indicate a different route to increase skeletal muscle GLUT4 expression, through the potential inhibition of the enzyme AICAR transformylase.

scholarly journals Control of glutamine synthesis in rat liver

1971 ◽  
Vol 124 (3) ◽  
pp. 653-660 ◽  
Author(s):  
P Lund
Keyword(s):  
Glutamine Synthetase ◽  
De Novo ◽  
Synthetic Medium ◽  
Linear Rate ◽  
Rapid Release ◽  
Initial Release ◽  
Glutamine Synthesis ◽  
Glutamine Production

1. On perfusion of livers from fed rats with a semi-synthetic medium containing no added amino acids there is a rapid release of glutamine during the first 15min (15.6±0.8μmol/h per g wet wt.; mean±s.e.m. of 35 experiments), followed by a low linear rate of production (3.6±0.3μmol/h per g wet wt.; mean±s.e.m. of three experiments). The rapid initial release can be accounted for as wash-out of preexisting intracellular glutamine. 2. Addition of readily permeating substrates, or substrate combinations, giving rise to intracellular glutamate or ammonia, or both, did not appreciably increase the rate of glutamine production over the endogenous rate. The maximum rate obtained was from proline plus alanine; even then the rate represented less than one-fortieth of the capacity of glutamine synthetase measured in vitro. 3. Complete inhibition of respiration in the perfusions [no erythrocytes in the medium; 1mm-cyanide; N2%CO2 (95:5) in the gas phase] or perfusion with glutamine synthetase inhibitors [l-methionine dl-sulphoximine; dl-(%)-allo-δ-hydroxylysine] abolishes the low linear rate of glutamine synthesis, but not the initial rapid release of glutamine. 4. In livers from 48h-starved rats initial release (0–15min) of glutamine was decreased (10.6±1.1μmol/h per g wet wt.; mean±s.e.m. of 11 experiments) and the subsequent rate of glutamine production was lower than in livers from fed rats. Again proline plus alanine was the only substrate combination giving an increase significantly above the endogenous rate. 5. The rate of glutamine synthesis de novo by the liver is apparently unrelated to the tissue content of glutamate or ammonia. 6. The blood glutamine concentration is increased by 50% within 1h of elimination of the liver from the circulation of rats in vivo. 7. There is an output of glutamine by the brain under normal conditions; the mean arterio-venous difference for six rats was 0.023μmol/ml. 8. The high potential activity of liver glutamine synthetase appears to be inhibited by some unknown mechanism: the function of the liver under normal conditions is probably the disposal of glutamine produced by extrahepatic tissues.

scholarly journals Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

2010 ◽  
Vol 192 (10) ◽  
pp. 2604-2612 ◽  
Author(s):  
Simon Klaffl ◽  
Bernhard J. Eikmanns
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Peptide Mass Fingerprinting ◽  
Transcriptional Analysis ◽  
Lysine Production ◽  
Ionization Time ◽  
Peptide Mass ◽  
Methyltransferase Gene ◽  
High Level

ABSTRACT Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for oxaloacetate of 1.4 mM and a V max of 201 μmol of oxaloacetate converted per min per mg of protein, resulting in a k cat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested.

scholarly journals Analysis of lines of mice selected for fat content: 3. Flux through the de novo lipid synthesis pathway

1991 ◽  
Vol 58 (2) ◽  
pp. 123-127 ◽  
Author(s):  
Emmanuel A. Asante ◽  
William G. Hill ◽  
Grahame Bulfield
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Lipid Synthesis ◽  
Fold Increase ◽  
Acid Synthesis ◽  
Fat Content ◽  
Direct Selection ◽  
Lean Line

SummaryThe flux through the de novo fatty acid synthesis pathway was estimated in lines of mice which differed substantially in fat content following 26 generations of selection at 10 weeks of age. Previous estimates of lipogenic enzyme activities had indicated an increase in the capacity for lipogenesis in the Fat compared to the Lean line. Therefore the in vivo flux in lipogenesis was measured in both liver and gonadal fat pad (GFP) tissues of males at 5 and 10 weeks of age, using the rat of incorporation of 3H from 3H2O and 14C from acetate and citra te into total lipids. AT both ages and in both tissues the Fat line had a higher flux, about 20% increase in the liver and up to three-fold increase (range 1·2- to 3·4-fold) in the GFP. We conclude that direct selection for fatness in mice has resulted in metabolic changes in the ratio of de novo fatty acid synthesis, and that the changes are largely detectable before 10 weeks, the age of selection.

scholarly journals Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells

1982 ◽  
Vol 2 (9) ◽  
pp. 1104-1114
Author(s):  
D L Gard ◽  
E Lazarides
Keyword(s):  
Cyclic Amp ◽  
Intermediate Filament ◽  
Specific Activity ◽  
Fold Increase ◽  
Major Protein ◽  
Myogenic Cells ◽  
Intermediate Filament Proteins ◽  
Dependent Protein

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

Rate of Incorporation of CO2 Carbon into Glucose and Other Body Constituents in Vivo

1975 ◽  
Vol 53 (5) ◽  
pp. 895-902 ◽  
Author(s):  
R. A. Shipley ◽  
A. P. Gibbons
Keyword(s):  
Fasting Glucose ◽  
Specific Activity ◽  
Fold Increase ◽  
Whole Body ◽  
Appreciable Amount ◽  
Hepatic Glycogen ◽  
Total Lipids ◽  
Fourfold Increase ◽  
Glucose Carbon

Specific activity curves of respired CO2 and of body glucose after intravenous NaH14CO3 as tracer and, in separate experiments, after [U-14C]glucose as tracer were employed to assess rate of interchange of carbon between HCO3 and glucose, and to calculate other rates of input and output for each of these substances. Solution for six rates attending the model was by integrals rather than by curve analysis. Fasting caused a twofold increase in rate of transport of CO2 carbon to glucose. Whereas in fed animals this rate was only 7% of the forward flow from glucose to CO2, it rose to 31% during fasting. Glucose carbon derived from CO2 rose from 3.7 to 20%. As expected, the rates of entry of new glucose to blood, and the conversion rate of glucose to products in body depots and to CO2 were reduced by fasting, whereas, the non-glucose input to CO2 was increased. Fasting was attended by a 20-fold increase in rate of conversion of CO2-derived carbon to hepatic glycogen and a fourfold increase to non-hepatic glycogen. Protein exceeded all whole-body depots for rate of acceptance of such carbon, and total lipids received an appreciable amount, but fasting caused no overall increase for either.

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