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Composition of the ornithine cycle in the conditioned media from cells irradiated with various doses of X-rays. Conditioned media from cultures with 50 000 cells were analysed for ornithine cycle compounds at 96 h following irradiation with 0–8 Gy X-rays (see Figure 4). Cell numbers in donor cultures at 96 h are included. Glutamine is shown for comparison.
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Cultured H35 hepatoma cells release a cytotoxic factor in response to irradiation with X-rays. When the conditioned medium from irradiated cells is given to nonirradiated cells, growth is inhibited and followed by cell death, possibly apoptosis, Analysis of the conditioned medium reveals a dramatic change in the ornithine (urea) cycle components af...
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A cytosolic cell-free system from rat liver containing the last three enzymes of the urea cycle, a number of cofactors and the substrates aspartate and citrulline was shown to synthesize urea at near-physiological rates ranging between 0.40 and 1.25 mumol/min per g of liver. This system was used to determine the kinetic parameters for arginase. Wit...
Skeletal muscle is known to contain arginase, but, because this enzyme is also present in erythrocytes, the exact origin of arginine-derived ornithine in peripheral tissues is uncertain. In the present studies, skeletal muscle cells obtained from regenerating hindlimb muscle of adult rats were grown in primary tissue culture for approximately 3 wk...
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Human recombinant arginase I cobalt coupled to polyethylene glycol 5000 (HuArg I [Co]-PEG5000) achieved potent in vitro depletion of arginine from tissue culture medium and cytotoxicity to many cancer cell lines. The recombinant enzyme also produced tumor growth inhibition of hepatocellular carcinoma and pancreatic carcinoma xenografts. Although th...
To determine the effect of trauma on arginase, an arginine-metabolizing enzyme, in cells of the immune system in humans.
Arginase, classically considered an enzyme exclusive to the liver, is now known to exist in cells of the immune system. Arginase expression is induced in these cells by cytokines interleukin (IL) 4, IL-10, and transforming growth...
The nitrogen excretory metabolism of the myxomycete Physarum polycephalum was studied. When cultured in partially defined broth medium or on agar, the principal excretory product was ammonia nitrogen. A small, variable quantity of urea was excreted in liquid culture. No uric acid or other purines were detected in the cultures. When microplasmodia w...
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... Many LAB from fermented foods such as kimchi, wine and cheese produce ornithine by the trans-formation of arginine via the arginine deiminase (ADI) pathway or the arginase-urease pathway (Arena et al., 1999;Kuensch et al., 1974;Liu et al., 2003). Accumulating data suggested that the transformation of arginine by these enzymes are cytotoxic to cancer cells (Ensor et al., 2002;Kim et al., 2009;Rijn et al., 2003). ...
... killed melanomas and hepatocellular carcinomas in vitro and in vivo (Ensor et al., 2002). Rijn et al. (2003) showed that the treatment of arginase inhibitor L-norvaline completely prevented the development of cytotoxicity in the X-ray irradiated H35 hepatoma cells. Thus, candidate products for the inhibitory effects could be ornithine cycle compounds and enzymes such as ornithine, citrulline, ammonia, ADI and arginase. ...
... The urea cycle is the final common pathway for the excretion of waste nitrogen in mammals and is the primary detoxification pathway for ammonia. 38 Ionizing radiation could lead to the ratio of urea to ammonia sharply dropping and thus give rise to hyperammonemia, 39,40 which suggests urea cycle defect development and further confirms our results. Since the urea cycle occurs mostly in the liver, the consistent reduction of the three urinary amino acid levels, including Arg, Orn and Cit, may reflect radiation induced liver damage to some extent. ...
Rapid radiation injury early triage after a radiological or nuclear exposure is vital for treatment of a large number of wounded people. Owing to the high-throughput analysis and minimally invasive nature of sample collection, radiation metabolomics has been recently applied to radiation damage research. In the present study, exploring the feasibility of estimating the acute radiation injury for early triage by means of urinary amino acid target analysis was attempted using a high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) technique combined with multivariate statistical analysis. The non-linear kernel partial least squares (KPLS) model was used to separate the control and different radiation dose groups. The classification of different groups was performed after feature selection instead of before feature selection, because of its better separation. The classification accuracy at various radiation injury levels at different time points (5, 24, 48 and 72 h) post-irradiation exposure was investigated. For most of the radiation damage levels, the classification accuracy at 72 h after exposure was superior to that at earlier time points. Additionally, the potential radiation injury biomarkers selected suggested that the urea cycle, glycine, serine and threonine metabolism, alanine, aspartate and glutamine metabolism and related metabolic pathways were involved. The findings suggest that non-invasive urinary biomarkers have great potential for serving as an effective tool for rapid triage of mass casualties in nuclear accidents and understanding the pathogenesis of radiation injury.
... For cell cycle analysis and Ki-67 immunocytochemistry experiments, HUVEC (5 × 10 5 cells/T-25 flask and 1 × 10 5 cells/slide, respectively) were synchronised by serum starving for 16 h. The medium was then replaced with fresh medium containing 4% serum with and without cytomix and 10 mM L-norvaline, a reported arginase inhibitor at millimolar concentration in macrophages and hepatoma cells, and cultures further incubated for 24 h19202122. ...
Objective. - Arginase is a nitric oxide synthase-alternative pathway for l-arginine breakdown leading to biosynthesis of urea and l-ornithine. Arginase pathway is inducible by inflammatory molecules-such as cytokines and bacterial endotoxin-in macrophages and smooth muscle cells. The presence of an arginase pathway in human endothelial cells and its possible modulation by inflammation are unknown. Methods. - We have: (i) characterised arginase pathway in terms of activity, isoform type and gene expression in a primary human umbilical vein endothelial cells (HUVEC) line; (ii) evaluated arginase functional role in cell proliferation with the aid of l-norvaline, an arginase inhibitor and (iii) determined the effects of tumour necrosis factor-alpha and endotoxin on arginase pathway. Results. - HUVEC showed a baseline arginase activity and expression of both arginase isoforms (arginase I and II (A-I and A-II, respectively)) which resulted in l-norvaline-inhibitable cellular polyamine synthesis. The baseline arginase activity is important for HUVEC proliferation as cell cycle analysis and nuclear factor Ki-67 immunostaining revealed. Following incubation with inflammatory molecules, arginase activity increased but HUVEC cell cycling decreased. Conclusions. - A-I and A-II are constitutively expressed in HUVEC where they take part to the regulation of cell cycling. Although arginase activity is positively modulated by inflammatory molecules, it is insufficient to counteract the overall cell cycling inhibiting effects of inflammation.
... In our previous report, we showed that the production of ammonia and the accompanying increases in ornithine and citrulline were significantly reduced by L-norvaline (Van Rijn et al, 2003). A similar result was obtained with L-agmatine, a natural derivative of arginine, although at a much lower dose of 0.2 mM (results not shown). ...
... Mycoplasma-contaminated and mycoplasma-free cell cultures were investigated as described earlier (Van Rijn et al, 2003). Concentrations are in mM. ...
The BJC is owned by Cancer Research UK, a charity dedicated to understanding the causes, prevention and treatment of cancer and to making sure that the best new treatments reach patients in the clinic as quickly as possible. The journal reflects these aims. It was founded more than fifty years ago and, from the start, its far-sighted mission was to encourage communication of the very best cancer research from laboratories and clinics in all countries. The breadth of its coverage, its editorial independence and it consistent high standards, have made BJC one of the world's premier general cancer journals. Its increasing popularity is reflected by a steadily rising impact factor.
The lung has raised significant concerns because of its radiosensitivity. Radiation-induced lung injury (RILI) has a serious impact on the quality of patients’ lives and limits the effect of radiotherapy on chest tumors. In clinical practice, effective drug intervention for RILI remains to be fully elucidated. Therefore, an in-depth understanding of the biological characteristics is essential to reveal the mechanisms underlying the complex biological processes and discover novel therapeutic targets in RILI. In this study, Wistar rats received 0, 10, 20 or 35 Gy whole-thorax irradiation (WTI). Lung and plasma samples were collected within 5 days post-irradiation. Then, these samples were processed using liquid chromatography–mass spectrometry (LC-MS). A panel of potential plasma metabolic markers was selected by correlation analysis between the lung tissue and plasma metabolic features, followed by the evaluation of radiation injury levels within 5 days following whole-thorax irradiation (WTI). In addition, the multiple metabolic dysregulations primarily involved amino acids, bile acids and lipid and fatty acid β-oxidation-related metabolites, implying disturbances in the urea cycle, intestinal flora metabolism and mitochondrial dysfunction. In particular, the accumulation of long-chain acylcarnitines (ACs) was observed as early as 2 d post-WTI by dynamic plasma metabolic data analysis. Our findings indicate that plasma metabolic markers have the potential for RILI assessment. These results reveal metabolic characteristics following WTI and provide new insights into therapeutic interventions for RILI.
There is a need for research to rapidly determine an individual's absorbed dose and its potential health effects after a potential radiological or nuclear event that could expose large portions of a population to ionizing radiation (IR). Studies on biomarker identification after radiation exposure could aid in biodosimetry, identifying individual dose absorbed, as well as biologic response, and administering immediate and proper medical care. Metabolomics on easily accessible biofluids is an emerging field with potential for high-throughput biodosimetry. While tremendous effort has been put into obtaining discovery based global radiation signatures from a number of biofluids and model organisms, quantitative targeted analysis on a subset of known radiation biomarkers is required to develop an optimized panel of biomarkers for future clinical applications. The current study analyzes levels of several known broad chemical groups (acylcarnitines, amino acids, phosphatidylcholines, and biogenic amines) affected by IR in serum from nonhuman primates (NHP) 7 days after exposure through multiple reaction monitoring (MRM) analysis with a tandem quadrupole mass spectrometry (MS) platform. We identified several novel metabolites affected by IR exposure through univariate and unsupervised multivariate analyses. Levels of acylcarnitines, amino acids, and phospholipids were perturbed indicating altered protein metabolism, fatty acid β-oxidation, and inflammation. Fold changes in carnitine and short-chain acylcarnitines (acetylcarnitine, propionylcarnitine, butyrylcarnitine, and valerylcarnitine) complement previous global radiation signatures on NHP; notably, the levels of change were lower than previously observed in urine. Decreased levels of glutamate, citrulline, and arginine after IR are biomarkers indicating gastrointestinal syndrome and perturbations to the urea cycle. Sex differences were also assessed and were more prevalent in circulating acylcarnitines and phospholipids after IR exposure. These biomarkers may be combined with previously described compounds from DNA damage to develop a defined metabolomic biodosimetry panel to be analyzed by MS platforms, which are increasingly available in clinical laboratories.
The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The Km and Vmax values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.
The objective of the present study was to identify a camptothecin (CPT) prodrug with optimal release and cytotoxicity properties for immobilization on a passively targeted microparticle delivery system. A series of alpha-amino acid ester prodrugs of CPT were synthesized, characterized, and evaluated. Four CPT prodrugs were synthesized with increasing aliphatic chain length (glycine (Gly) (2a), alanine (Ala) (2b), aminobutyric acid (Abu) (2c), and norvaline (Nva) (2d)). Prodrug reconversion was studied at pH 6.6, 7.0, and 7.4 corresponding to tumor, lung, and extracellular/physiological pH, respectively. Cytotoxicity was evaluated in A549 human lung carcinoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hydrolytic reconversion rate to parent CPT increased with decreasing side chain length as well as increasing pH. The Hill slope of 2d was significantly less than CPT and the other prodrugs tested, indicating a higher cell death rate at lower concentrations. These results suggest that 2d is the best candidate for a passively targeted sustained release lung delivery system.
Interest has recently been revived in enzymes that degrade essential amino acids. Arginine-catabolizing enzymes now predominate and are discussed in this review. Apart from reducing tumor load through cell death occurring as a result of deprivation alone, these catabolic enzymes conveniently leave the remaining malignant cells vulnerable to other therapeutic modalities through combinatorial treatments with cycle-dependent drugs, the timing of additional treatment after deprivation being crucial.
