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Cells Aug 2021Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that...
Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.
Topics: Arginase; Arginine; Case-Control Studies; Cell Culture Techniques; Cells, Cultured; Collagen; Collagen Type I; Cornea; Extracellular Matrix; Fibroblasts; Humans; Keratoconus; Nitric Oxide Synthase; Ornithine; Spermidine; Up-Regulation
PubMed: 34440845
DOI: 10.3390/cells10082076 -
PloS One 2018Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To...
Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidylethanolamine lipids plus cholesterol. Niosomes are highly stable and only slightly more leaky than liposomes as assayed by calcein leakage; the permeability for ions (KCl) is higher than that of liposomes. Contrary to liposomes, the size of niosomes decreases substantially upon freezing in liquid nitrogen and subsequent thawing, as shown by cryo-EM and dynamic light scattering. The packing of niosomal membranes was determined by laurdan fluorescence and is slightly lower than that of liposomes. We did not succeed in the functional reconstitution of the L-arginine/L-ornithine antiporter ArcD2 in niosomes, which we attribute to the non-ionic nature of the surfactants. The antimicrobial peptides alamethicin and melittin act similarly on niosomes and liposomes composed of unsaturated components, whereas both niosomes and liposomes are unaffected when saturated amphiphiles are used. In conclusion, in terms of stability and permeability for drug-size molecules niosomes are comparable to liposomes and they may offer an excellent, inexpensive alternative for delivery purposes.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Alamethicin; Antimicrobial Cationic Peptides; Arginine; Cholesterol; Cryoelectron Microscopy; Detergents; Drug Delivery Systems; Fluoresceins; Hexoses; Light; Lipids; Liposomes; Melitten; Nitrogen; Ornithine; Osmosis; Permeability; Phosphatidylethanolamines; Polysorbates; Scattering, Radiation; Surface-Active Agents
PubMed: 29649223
DOI: 10.1371/journal.pone.0194179 -
Molecules (Basel, Switzerland) Apr 2021Derivatization of amino acids by 2 M HCl/CHOH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in...
GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of -Carboxy-ornithine and -Carboxy-lysine.
Derivatization of amino acids by 2 M HCl/CHOH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)-(PFP). This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (dMe)-(PFP), from synthetic amino acids and 2 M HCl/CDOD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be -Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being -Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (,)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP) and hCit-(PFP) are esterified on their -Carboxylic groups and on their activated groups. Procedure B also allows in situ preparation of (dMe)-(PFP) from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.
Topics: Amino Acids; Citrulline; Fluorocarbons; Gas Chromatography-Mass Spectrometry; Lysine; Ornithine
PubMed: 33921162
DOI: 10.3390/molecules26082301 -
The Journal of Biological Chemistry Nov 2018Cancer is a set of diseases characterized by uncontrolled cell growth. In certain cancers of the gastrointestinal tract, the adenomatous polyposis coli (APC) tumor... (Review)
Review
Cancer is a set of diseases characterized by uncontrolled cell growth. In certain cancers of the gastrointestinal tract, the adenomatous polyposis coli (APC) tumor suppressor gene is altered in either germline or somatic cells and causes formation of risk factors, such as benign colonic or intestinal neoplasia, which can progress to invasive cancer. APC is a key component of the WNT pathway, contributing to normal GI tract development, and APC alteration results in dysregulation of the pathway for production of polyamines, which are ubiquitous cations essential for cell growth. Studies with mice have identified nonsteroidal anti-inflammatory drugs (NSAIDs) and difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, as potent inhibitors of colon carcinogenesis. Moreover, gene expression profiling has uncovered that NSAIDs activate polyamine catabolism and export. Several DFMO-NSAID combination strategies are effective and safe methods for reducing risk factors in clinical trials with patients having genetic or sporadic risk of colon cancer. These strategies affect cancer stem cells, inflammation, immune surveillance, and the microbiome. Pharmacotherapies consisting of drug combinations targeting the polyamine pathway provide a complementary approach to surgery and cytotoxic cancer treatments for treating patients with cancer risk factors. In this Minireview, we discuss the role of polyamines in colon cancer and highlight the mechanisms of select pharmacoprevention agents to delay or prevent carcinogenesis in humans.
Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Chemoprevention; Colonic Neoplasms; Eflornithine; Humans; Polyamines
PubMed: 30355737
DOI: 10.1074/jbc.TM118.003343 -
Fluids and Barriers of the CNS Feb 2022In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB)...
BACKGROUND
In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by L-ornithine. Our goal was also to identify whether L-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes.
METHODS
AP was induced in rats by the intraperitoneal administration of L-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of L-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of L-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, β-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry.
RESULTS
In the L-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. L-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and β-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in L-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after L-ornithine treatment.
CONCLUSION
We demonstrated BBB damage in the L-ornithine-induced rat AP model suggesting a general, AP model independent effect. L-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic L-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.
Topics: Acute Disease; Animals; Blood-Brain Barrier; Brain; Cells, Cultured; Endothelial Cells; Endothelium; Ornithine; Pancreatitis; Rats; Tight Junctions
PubMed: 35177109
DOI: 10.1186/s12987-022-00308-0 -
Comparative Biochemistry and... Mar 2020Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein...
Supplementation of arginine, ornithine and citrulline in rainbow trout (Oncorhynchus mykiss): Effects on growth, amino acid levels in plasma and gene expression responses in liver tissue.
Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3 h and 24 h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24 h and 3 h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3 h and 24 h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3 h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.
Topics: Amino Acids; Animals; Arginine; Citrulline; Dietary Supplements; Liver; Oncorhynchus mykiss; Ornithine
PubMed: 31812671
DOI: 10.1016/j.cbpa.2019.110632 -
Clinical Journal of the American... Dec 2020Genetic variants in , a liver- and kidney-specific acetyltransferase encoding gene, have been associated with eGFR and CKD in European populations. Higher circulating...
BACKGROUND AND OBJECTIVES
Genetic variants in , a liver- and kidney-specific acetyltransferase encoding gene, have been associated with eGFR and CKD in European populations. Higher circulating levels of two -associated metabolites, N--acetylornithine and N-acetyl-1-methylhistidine, have been linked to lower eGFR and higher risk of incident CKD in the Black population. We aimed to expand upon prior studies to investigate associations between rs13538, a missense variant in , N-acetylated amino acids, and kidney failure in multiple, well-characterized cohorts.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS
We conducted analyses among participants with genetic and/or serum metabolomic data in the African American Study of Kidney Disease and Hypertension (AASK; =962), the Atherosclerosis Risk in Communities (ARIC) study (=1050), and Bio, an electronic health record-linked biorepository (=680). Separately, we evaluated associations between rs13538, urinary N-acetylated amino acids, and kidney failure in participants in the German CKD (GCKD) study (=1624).
RESULTS
Of 31 N-acetylated amino acids evaluated, the circulating and urinary levels of 14 were associated with rs13538 (<0.05/31). Higher circulating levels of five of these N-acetylated amino acids, namely, N--acetylornithine, N-acetyl-1-methylhistidine, N-acetyl-3-methylhistidine, N-acetylhistidine, and N2,N5-diacetylornithine, were associated with kidney failure, after adjustment for confounders and combining results in meta-analysis (combined hazard ratios per two-fold higher amino acid levels: 1.48, 1.44, 1.21, 1.65, and 1.41, respectively; 95% confidence intervals: 1.21 to 1.81, 1.22 to 1.70, 1.08 to 1.37, 1.29 to 2.10, and 1.17 to 1.71, respectively; all values <0.05/14). None of the urinary levels of these N-acetylated amino acids were associated with kidney failure in the GCKD study.
CONCLUSIONS
We demonstrate significant associations between an gene variant and 14 N-acetylated amino acids, five of which had circulation levels that were associated with kidney failure.
Topics: Acetylation; Acetyltransferases; Adult; Aged; Aged, 80 and over; Amino Acids; Black People; Disease Progression; Female; Follow-Up Studies; Glomerular Filtration Rate; Histidine; Humans; Kidney Failure, Chronic; Male; Metabolomics; Methylhistidines; Middle Aged; Ornithine; Polymorphism, Single Nucleotide; Prospective Studies; Randomized Controlled Trials as Topic; White People
PubMed: 33380473
DOI: 10.2215/CJN.08600520 -
Clinical Nutrition (Edinburgh, Scotland) Dec 2022The interplay among dietary intake, gut microbiota, gut metabolites and circulating metabolites in adolescents is barely known, not to mention sex-dependent pattern. We...
BACKGROUND & AIMS
The interplay among dietary intake, gut microbiota, gut metabolites and circulating metabolites in adolescents is barely known, not to mention sex-dependent pattern. We aimed to explore unique profiles of gut bacterial, gut metabolites and circulating metabolites from both genders of adolescents due to BMI and eating pattern.
METHODS
Clinical indices, fecal gut microbiota, fecal and plasma metabolites, and diet intake information were collected in case-control sample matched for normal and obesity in girls (normal = 12, obesity = 12) and boys (normal = 20, obesity = 20), respectively. 16S rRNA gene sequencing and untargeted metabolomics was performed to analysis the signature of gut microbiota and metabolites. Unique profiles of girls associated with BMI and eating pattern was revealed by Spearman's correlations analysis, co-occurrence network analysis, Kruskal-Wallis test, and Wilcoxon rank-sum test.
RESULTS
Gender difference was found between normal and obese adolescents in gut microbiota, fecal metabolites, and plasma metabolites. The Parabacteroides were only decreased in obese girls. And the characteristic of obese girls' and boys' cases in fecal and plasma was xanthine and glutamine, ornithine and LCA, respectively. Soy products intake was negatively associated with Parabacteroides. The predicted model has a higher accuracy based on the combined markers in obesity boys (AUC = 0.97) and girls (AUC = 0.97), respectively.
CONCLUSIONS
Reduced abundance of Phascolarctobacterium and Parabacteroides, as well as the increased fecal xanthine and ornithine, may provide a novel biomarker signature in obesity girls and boys. Soy products intake was positively and negatively associated with Romboutsia and Parabacteroides abundance, respectively. And the combined markers facilitate the accuracy of predicting obesity in girls and boys in advance.
Topics: Adolescent; Humans; Female; Male; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Pediatric Obesity; Feces; Metabolome; Eating; Biomarkers; Ornithine; Xanthines
PubMed: 36351362
DOI: 10.1016/j.clnu.2022.10.009 -
FEBS Letters Dec 2021The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified...
The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (k /K ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.
Topics: Bacterial Proteins; Cysteine; Enzyme Stability; Glutamic Acid; Kinetics; Ornithine; Serine; Substrate Specificity; Thermotoga maritima; Transaminases
PubMed: 34747014
DOI: 10.1002/1873-3468.14222 -
Biochimie Dec 2020One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene...
One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene expression. However, what AGEs exist in vivo and how individual AGEs are produced and impact body metabolic process to cause diabetes complications are not understood. We developed a new precise method to measure AGEs using LC-MS/MS with a new column and measured 7 free AGEs, including N(6)-carboxymethyllysine (CML), N(6)-(1-carboxyethyl)-l-lysine (CEL) and N5-(5-hydro-5-methyl-4-imidazolon-2-yl)L-ornithine (MG-H1), in human blood components. Blood was obtained from 9 people, and free AGEs were measured in individual blood components with LC-MS/MS before and after a meal. Free CML and CEL were abundant in erythrocytes, with 92% of free CML and 85% of free CEL localized in erythrocytes. In contrast, 60% of free MG-H1 was distributed in the serum. After the meal, free serum MG-H1 increased, but CML and CEL did not. CML and CEL are mainly distributed in erythrocytes and were not affected by the meal, indicating that they are produced in vivo. However, the main source of MG-H1 is the meal. The effect of genetic polymorphisms on AGEs was also investigated. Low activity type aldehyde dehydrogenase 2 (ALDH2) increased the CML concentration in the blood. This is the first observation that shows that the metabolic process of CML and CEL is different from that of MG-H1 and the effect of ALDH2 SNPs on CML.
Topics: Adult; Alcohol Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Chromatography, High Pressure Liquid; Erythrocytes; Female; Glycation End Products, Advanced; Healthy Volunteers; Humans; Lysine; Male; Meals; Middle Aged; Ornithine; Polymorphism, Single Nucleotide; Tandem Mass Spectrometry; Young Adult
PubMed: 32946992
DOI: 10.1016/j.biochi.2020.09.010