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Plant Physiology May 2021
Topics: Apoptosis; Cell Membrane; Cucumis sativus; Fruit; Heat-Shock Proteins; Magnoliopsida; Ornithine Decarboxylase; Plant Necrosis and Chlorosis; Pollination; Sphingolipids; Xylem
PubMed: 35237801
DOI: 10.1093/plphys/kiab104 -
Cancer Medicine May 2024Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in...
BACKGROUND
Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses.
METHODS
We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression in vitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 μM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05.
RESULTS
There was not a significant loss of cell viability or gain of apoptotic activity in the in vitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO.
CONCLUSION
DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.
Topics: Neuroblastoma; Humans; Animals; Cell Line, Tumor; Mice; Apoptosis; Eflornithine; Xenograft Model Antitumor Assays; Cell Survival; Carcinogenesis; Cell Cycle; Cell Proliferation; Antineoplastic Agents; Female
PubMed: 38686627
DOI: 10.1002/cam4.7207 -
Frontiers in Bioengineering and... 2019L-ornithine, a valuable non-protein amino acid, has a wide range of applications in the pharmaceutical and food industries. Currently, microbial fermentation is a... (Review)
Review
L-ornithine, a valuable non-protein amino acid, has a wide range of applications in the pharmaceutical and food industries. Currently, microbial fermentation is a promising, sustainable, and environment-friendly method to produce L-ornithine. However, the industrial production capacity of L-ornithine by microbial fermentation is low and rarely meets the market demands. Various strategies have been employed to improve the L-ornithine production titers in the model strain, , which serves as a major indicator for improving the cost-effectiveness of L-ornithine production by microbial fermentation. This review focuses on the development of high L-ornithine-producing strains by metabolic engineering and reviews the recent advances in breeding strategies, such as reducing by-product formation, improving the supplementation of precursor glutamate, releasing negative regulation and negative feedback inhibition, increasing the supply of intracellular cofactors, modulating the central metabolic pathway, enhancing the transport system, and adaptive evolution for improving L-ornithine production.
PubMed: 31998705
DOI: 10.3389/fbioe.2019.00440 -
The FEBS Journal Jan 2021In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine...
In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the K and V values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter.
Topics: Adenosine Triphosphate; Amino Acid Transport Systems; Ammonia; Antiporters; Arginine; Bacterial Proteins; Carbon Dioxide; Energy Metabolism; Gene Expression Regulation, Bacterial; Hydrolases; Kinetics; Lactococcus lactis; Liposomes; Ornithine; Ornithine Carbamoyltransferase; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphotransferases (Carboxyl Group Acceptor); Recombinant Proteins
PubMed: 32306469
DOI: 10.1111/febs.15337 -
Acta Medica (Hradec Kralove) 2022To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim...
INTRODUCTION
To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function.
METHODS
We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors).
RESULTS
A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 μmol/L; median 63 μmol/L, IQR 34 μmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group.
CONCLUSIONS
Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.
Topics: Male; Humans; Female; Adult; Middle Aged; Aged; Citrulline; Celiac Disease; Ornithine; Diet
PubMed: 36735884
DOI: 10.14712/18059694.2022.22 -
Advances in Nutrition (Bethesda, Md.) Jul 2023Individual amino acids are widely popular as supplements because of various perceived and real health benefits. However, currently, there are no recommendations set by... (Review)
Review
Individual amino acids are widely popular as supplements because of various perceived and real health benefits. However, currently, there are no recommendations set by national health agencies for tolerable upper intake levels (UL) for amino acids because of a lack of well-conducted human dose-response trials. In the past decade, under the initiative of the International Council on Amino Acid Science, a nonprofit organization, a series of UL human clinical studies were conducted. The goal of this narrative review is to summarize the studies on 6 essential amino acids (leucine, tryptophan, methionine, lysine, histidine, and phenylalanine), 2 nonessential amino acids (arginine and serine), and 2 nonproteinogenic amino acids (ornithine and citrulline) and provide the first set of ULs. A brief background of the concept of the DRI framework of UL, the concept of UL for amino acids, and a perspective of the results are also provided. The data suggest that in relatively healthy adult individuals, the tested amino acids are well tolerated, and ULs, or the no-observed-adverse-effect-level (NOAEL), lowest-observed-adverse-effect-level (LOAEL), can be determined. The ULs were for leucine-young (35 g/d), tryptophan (4.5 g/d), and leucine-elderly (30 g/d); NOAEL and LOAEL for methionine at 3.2 and 6.4 g/d, respectively; NOAEL for arginine (30 g/d); NOAEL and LOAEL for lysine at 6 and 7.5 g/d, respectively; NOAEL and LOAEL for histidine at 8 and 12 g/d, respectively; and NOAEL for phenylalanine (12 g/d), serine (12 g/d), ornithine (12 g/d) and citrulline (24 g/d). This first set of human UL data are hoped to help national and international agencies set safety standards for supplemental amino acids.
Topics: Humans; Aged; Leucine; Tryptophan; Lysine; Histidine; Citrulline; Methionine; Arginine; Phenylalanine; Serine; Ornithine
PubMed: 37062432
DOI: 10.1016/j.advnut.2023.04.004 -
Metabolites Dec 2020Fatty Acyl esters of Hydroxy Fatty Acids (FAHFA) encompass three different lipid families which have incorrectly been classified as wax esters. These families include... (Review)
Review
Fatty Acyl esters of Hydroxy Fatty Acids (FAHFA) encompass three different lipid families which have incorrectly been classified as wax esters. These families include (i) Branched-chain FAHFAs, involved in the regulation of glucose metabolism and inflammation, with acylation of an internal branched-chain hydroxy-palmitic or -stearic acid; (ii) ω-FAHFAs, which function as biosurfactants in a number of biofluids, are formed via acylation of the ω-hydroxyl group of very-long-chain fatty acids (these lipids have also been designated as o-acyl hydroxy fatty acids; OAHFA); and (iii) Ornithine-FAHFAs are bacterial lipids formed by the acylation of short-chain 3-hydroxy fatty acids and the addition of ornithine to the free carboxy group of the hydroxy fatty acid. The differences in biosynthetic pathways and cellular functions of these lipid families will be reviewed and compared to wax esters, which are formed by the acylation of a fatty alcohol, not a hydroxy fatty acid. In summary, FAHFA lipid families are both unique and complex in their biosynthesis and their biological actions. We have only evaluated the tip of the iceberg and much more exciting research is required to understand these lipids in health and disease.
PubMed: 33348554
DOI: 10.3390/metabo10120512 -
SLAS Discovery : Advancing Life... Oct 2020Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the...
Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.
Topics: Arginase; Arginine; Enzyme Inhibitors; Fluorescence; High-Throughput Screening Assays; Humans; Neoplasms; Ornithine; T-Lymphocytes
PubMed: 32418491
DOI: 10.1177/2472555220919340 -
Proceedings of the National Academy of... Dec 2020Recessive loss-of-function mutations in () are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that...
Recessive loss-of-function mutations in () are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a strain deficient in the ATP13A2 ortholog These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by , the ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.
Topics: Activating Transcription Factor 4; Adenosine Triphosphatases; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Eflornithine; Fibroblasts; Lysosomes; Mitochondria; Mutation; Oxidative Stress; Parkinson Disease; Polyamines; Proton-Translocating ATPases; Rotenone; Spermine; Transcription Factor CHOP; Transcription Factors
PubMed: 33229544
DOI: 10.1073/pnas.1922342117 -
Journal of Food Protection Feb 2021Shewanella baltica, one of the dominant spoilers of seafoods, can synthesize putrescine from ornithine under acidic conditions, which could result in food spoilage and...
ABSTRACT
Shewanella baltica, one of the dominant spoilers of seafoods, can synthesize putrescine from ornithine under acidic conditions, which could result in food spoilage and health problems. We identified three regulatory enzymes (SpeC, SpeF, and PotE) in the ornithine decarboxylation (ODC) pathway of S. baltica by searching the NCBI database and exploring their functional roles through gene knock-out technology. The ornithine decarboxylase SpeC is an auxiliary adjustor of the ODC system, whereas the ornithine-putrescine transporter SpeE and ornithine decarboxylase SpeF participate in the production of extracellular putrescine. Exogenous addition of ornithine and putrescine promotes the extracellular secretion of putrescine by upregulating the expression of speF and potE. The putrescine biosynthesis and alkalization of cytoplasm is enhanced at weak acidic pH compared with neutral pH, especially at pH 6.0. The maximum upregulation of ODC genes and the optimum decarboxylation activity of SpeF are achieved in a weak acidic environment (pH 6.0), suggesting that the ODC pathway plays an important role in putrescine production and the cytoplasmic acid counteraction of S. baltica. This study contributes to a wider understanding of spoilage mechanisms in food systems and provides theoretical support for developing novel seafood preservation methods.
Topics: Decarboxylation; Ornithine; Putrescine; Shewanella
PubMed: 33003195
DOI: 10.4315/JFP-20-227