3-tyrosine and 2-tyrosine

Concomitant tumor resistance (CR) is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although previous studies indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In a recently published study, we identified this factor as meta-tyrosine and ortho-tyrosine, 2 isomers of tyrosine that would not be present in normal proteins. In 3 different murine models of cancer that generate CR, both meta- and ortho-tyrosine inhibited tumor growth. Additionally, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isomers were mediated in part by early inhibition of the MAP/ERK pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy in G(0)-phase. Other mechanisms, putatively involving the activation of an intra-S-phase checkpoint, would also inhibit tumor proliferation by accumulating cells in S-phase. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors or after other stressors that may promote the escape of metastases from dormancy, an issue that is of pivotal importance to oncologists and their patients.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Mice; Neoplasm Metastasis; Neoplasms; S Phase; STAT3 Transcription Factor; Tyrosine

We studied the effect of short-term nadolol administration on the reactive oxygen species (ROS) generation by polymorphonuclear leukocytes and mononuclear cells in 8 normal subjects. At a oral dose of 40 mg/day for 5 days, nadolol produced a decrease in the ROS generation by leukocytes. ROS generation by polymorphonuclear leukocytes decreased by 38% from 134 +/- 44 mV at baseline to 83 +/- 34 mV after 5 days (p = 0.005), and ROS generation by mononuclear cells decreased by 33% from 174 +/- 69 mV at baseline to 117 +/- 55 mV after 5 days (p = 0.015). There was also a significant reduction in linoleic acid oxidation as reflected by the lower levels of 9- and 13- hydroxy-octadecadienoic acid after 5 days. There was no change in the plasma thiobarbituric acid-reacting substances, a less sensitive index of oxidative damage to lipids. There was also no significant change in the levels of metatyrosine and orthotyrosine, which are known indexes of oxidative damage to amino acids and proteins. The absence of a significant change in metatyrosine, orthotyrosine, and thiobarbituric acid-reacting substances may reflect the short duration of nadolol administration and the decreased ROS load. Because ROS may induce lipid peroxidation, this inhibitory effect of nadolol on ROS generation by leukocytes and linoleic acid oxidation may inhibit low-density lipoprotein oxidation and thus atherogenesis. This effect may partly explain the favorable outcomes observed in patients with coronary artery disease on long-term beta-blocker therapy.

Topics: Administration, Oral; Adrenergic beta-Antagonists; Adult; Chromatography, High Pressure Liquid; Female; Humans; Leukocytes, Mononuclear; Linoleic Acid; Male; Nadolol; Neutrophils; Oxidation-Reduction; Phenylalanine; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Tyrosine

In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention.

Topics: Animals; Antibodies, Monoclonal; Catalytic Domain; CHO Cells; Cricetulus; Female; Leucine; Models, Molecular; Phenylalanine-tRNA Ligase; Protein Conformation; Recombinant Proteins; Tyrosine

To identify the irradiated meats, various parameters that affect extraction efficiency of tyrosine positional isomers were evaluated. The optimum procedure employed simple extraction by 0.1% formic acid and protein precipitation by acetone. Baseline separation for the extract was carried out on LC-fluorescence detection (FLD) and LC-tandem mass spectrometry (MS/MS). The LC-FLD and LC-MS/MS method had LOD of 1.7-2.1 and 0.3-0.5 ng/mL, respectively, and showed excellent linear correlation over three orders of magnitude, obtained ideal recoveries (78.68-88.90%) and RSD (≤ 8%). The methods were successfully applied in multiple samples. For o- and m-tyrosine, the order of descending trend was: chicken>beef > hairtail > pork and chicken > hairtail > beef > pork, respectively. The radiation dose could be quantitatively evaluated by the nonlinear correlation (y = A(0) x (2) + A(1)x + A(2)) with coefficients of determination r(2) > 0.998 in individual meat samples.

Topics: Acetone; Animals; Cattle; Chickens; Chromatography, Liquid; Dose-Response Relationship, Radiation; Fluorescence; Food Irradiation; Linear Models; Meat; Proteins; Reproducibility of Results; Swine; Tandem Mass Spectrometry; Tyrosine

Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients.

Topics: Animals; Chromatography, High Pressure Liquid; Disease Resistance; Extracellular Signal-Regulated MAP Kinases; Female; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Phenylalanine; STAT3 Transcription Factor; Tyrosine

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.

Topics: Adolescent; Adult; Antioxidants; beta-Thalassemia; Bilirubin; Biomarkers; Female; Humans; Iron; Lipid Peroxidation; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress; Proteins; Tyrosine

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentrat

Topics: Aged; Cataract; Cross-Sectional Studies; Diabetes Mellitus, Type 2; Dihydroxyphenylalanine; Eye Proteins; Humans; Hydroxyl Radical; Lens, Crystalline; Middle Aged; Phenylalanine; Solubility; Tyrosine; Water

Recent evidence argues strongly that the marked increase in risk for atherosclerotic heart disease seen in diabetics cannot be explained by a generalized increase in oxidative stress. Here, we used streptozotocin to induce hyperglycemia in cynomolgus monkeys for 6 months and tested whether high glucose levels promote localized oxidative damage to artery wall proteins. We focused on three potential agents of oxidative damage: hydroxyl radical, tyrosyl radical, and reactive nitrogen species. To determine which pathways operate in vivo, we quantified four stable end products of these reactants -- ortho-tyrosine, meta-tyrosine, o,o'-dityrosine, and 3-nitrotyrosine -- in aortic proteins. Levels of ortho-tyrosine, meta-tyrosine, and o,o'-dityrosine, but not of 3-nitrotyrosine, were significantly higher in aortic tissue of hyperglycemic animals. Of the oxidative agents we tested, only hydroxyl radical mimicked this pattern of oxidized amino acids. Moreover, tissue levels of ortho-tyrosine and meta-tyrosine correlated strongly with serum levels of glycated hemoglobin, a measure of glycemic control. We conclude that short-term hyperglycemia in primates promotes oxidation of artery wall proteins by a species that resembles hydroxyl radical. Our observations suggest that glycoxidation reactions in the arterial microenvironment contribute to early diabetic vascular disease, raising the possibility that antioxidant therapies might interrupt this process.

Topics: Animals; Aorta; Arteriosclerosis; Diabetes Mellitus, Experimental; Glucose; Glycated Hemoglobin; Hydroxyl Radical; Lipids; Macaca fascicularis; Male; Mass Spectrometry; Oxidation-Reduction; Time Factors; Tyrosine

ABSTRACT Since glucose intake acutely increases reactive oxygen species (ROS) generation by polymorphonuclear leucocytes (PMN) and mononuclear cells (MNC), we have now investigated whether a fast over a period of 48h reduces ROS generation by these cells. Eight normal subjects were fasted for 48h. Blood samples were obtained at 0, 24h and 48h. ROS generation by PMN fell significantly at 24h (66.1 +/- 19.5% of basal) and further at 48h (45.9 +/- 23.0 % of basal; p < 0.001). ROS generation by MNC fell to 62.4 +/- 16.5% at 24h and by 48.4 +/- 16.5% (p < 0.001) by 48h. The level of p47(phox) subunit, an index of NADPH oxidase, the enzyme converting molecular oxygen to superoxide (O(.)(2)(-)) radical, also fell in parallel. Plasma o-tyrosine/phenylalanine ratio fell significantly from 0.326 +/- 0.053 mmol/mol to 0.303 +/- 0.055 mmol/mol at 48h and m-tyrosine/phenylalanine ratio fell from 0.363 +/- 0.063 mmol/mol to 0.340 +/- 0.064 mmol/mol (p < 0.05). Thus, a 48h fast may reduce ROS generation, total oxidative load and oxidative damage to amino acids.

Topics: Adult; Fasting; Humans; Middle Aged; Monocytes; NADPH Oxidases; Neutrophils; Osmolar Concentration; Phenylalanine; Phosphoproteins; Reactive Oxygen Species; Time Factors; Tyrosine

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

Topics: Aging; Animals; Brain; Energy Metabolism; Female; Free Radicals; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Oxidative Phosphorylation; Proteins; Rats; Rats, Wistar; Tyrosine

The purpose of this study was to test whether carvedilol has an antioxidant effect in humans in vivo.. We administered 3.125 mg of carvedilol twice daily to normal subjects for 1 week. ROS generation by polymorphonuclear leukocytes and mononuclear cells fell from 314+/-183.43 and 303+/-116 mV to 185+/-157 and 189+/-63 mV (P<0.025), respectively. m-Tyrosine fell from 4.24+/-0.99 to 4.03+/-0.97 ng/mL (P=0.01), and o-tyrosine fell from 4.59+/-1.10 to 4.24+/-0.99 ng/mL (P=0.004) in the absence of a change in phenylalanine concentrations.. We conclude that carvedilol significantly inhibits ROS generation by leukocytes and oxidative conversion of phenylalanine to m- and o-tyrosine.

Topics: Adult; Antioxidants; Carbazoles; Carvedilol; Humans; Monocytes; Neutrophils; Oxidation-Reduction; Phenylalanine; Propanolamines; Reactive Oxygen Species; Tyrosine

We have examined the effect of short-term triiodothyronine (T3) administration on reactive oxygen species (ROS) generation by leukocytes in 9 euthyroid subjects. At a dose of 60 microg/d orally for 7 days, T3 induced a significant increase in ROS generation by mononuclear cells (MNCs) from 183 +/- 102 mV at baseline to 313 +/- 111 mV on the seventh day (P < .02), and by polymorphonuclear leukocytes (PMNLs) from 195 +/- 94 mV at baseline to 302 +/- 104 mV on the seventh day (P < .02). There was also a significant increase in meta-tyrosine (P < .001) and ortho-tyrosine (P < .001), known indices of oxidative damage to proteins and amino acids. However, there was no increase in plasma thiobarbituric acid-reactive substances (TBARS), an index of oxidative damage to lipids, and in the level of carbonylated proteins, a less sensitive index to assess protein oxidation. There was no decrease in the level of antioxidants such as alpha-tocopherol, vitamin A, beta-carotene, lycopene, and lutein/zeaxanthin. The stimulatory effect on ROS generation may reflect a generalized increase in metabolic activity or may be a specific effect on NADPH oxidase in leukocyte membranes. The absence of a significant change in TBARS, carbonylated proteins, alpha-tocopherol, vitamin A, beta-carotene, lycopene, and lutein/zeaxanthin may reflect the short duration of the increased ROS load.

Topics: Adult; Antioxidants; Female; Humans; Leukocytes; Male; NADPH Oxidases; Oxidative Stress; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Thyroid Function Tests; Triiodothyronine; Tyrosine

Topics: Animals; Biomarkers; Brain Chemistry; Humans; Isomerism; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Neuron Disease; Muscle, Skeletal; Spinal Cord; Superoxide Dismutase; Tyrosine

Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors may have therapeutic potential as psychotropic agents. A series of mononitro- and dinitro-2- and 3-hydroxyphenylalanines was prepared, and their activity compared with willardiine, 5-nitrowillardiine, AMPA, and 2,4,5-trihydroxyphenylalanine (6-hydroxydopa) as inhibitors of specific [3H]AMPA and [3H]kainate binding in rat brain homogenates. The most active compounds were highly acidic (pKa 3-4), namely, 2-hydroxy-3,5-dinitro-DL-phenylalanine (13; [3H]AMPA IC50 approximately equal to 25 microM) and 3-hydroxy-2,4-dinitro-DL-phenylalanine (19; [3H]AMPA IC50 approximately equal to 5 microM). Two other dinitro-3-hydroxyphenylalanines, and 3,5-dinitro-DL-tyrosine, were considerably less active. Various mononitrohydroxyphenylalanines, which are less acidic, were also less active or inactive, and 2- and 3-hydroxyphenylalanine (o- and m-tyrosine) were inactive. Compounds 13 and 19, DL-willardiine (pKa 9.3, [3H]AMPA IC50 = 2 microM), and 5-nitro-DL-willardiine (pKa 6.4, [3H]AMPA IC50 = 0.2 microM) displayed AMPA >> kainate selectivity in binding studies. Compound 19 was an AMPA-like agonist, but 13 was an antagonist in an AMPA-evoked norepinephrine release assay in rat hippocampal nerve endings. Also, compound 13 injected into the rat ventral pallidum antagonized the locomotor activity elicited by systemic amphetamine.

Topics: Animals; Binding, Competitive; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Isomerism; Models, Chemical; N-Methylaspartate; Phencyclidine; Quinoxalines; Radioligand Assay; Rats; Receptors, AMPA; Structure-Activity Relationship; Tyrosine

The habit of betel quid chewing, common in South-East Asia and the South Pacific islands, is causally associated with an increased risk of oral cancer. Reactive oxygen species formed from polyphenolic betel quid ingredients and lime at alkaline pH have been implicated as the agents responsible for DNA and tissue damage. To determine whether hydroxyl radical (HO.) is generated in the human oral cavity during chewing of betel quid, the formation of o- and m-tyrosine from L-phenylalanine was measured. Both o- and m-tyrosine were formed in vitro in the presence of extracts of areca nut and/or catechu, transition metal ions such as Cu2+ and Fe2+ and lime or sodium carbonate (alkaline pH). Omission of any of these ingredients from the reaction mixture significantly reduced the yield of tyrosines. Hydroxyl radical scavengers such as ethanol, D-mannitol and dimethylsulfoxide inhibited the phenylalanine oxidation in a dose-dependent fashion. Five volunteers chewed betel quid consisting of betel leaf, areca nut, catechu and slaked lime (without tobacco). Their saliva, collected after chewing betel quid, contained high concentrations of p-tyrosine, but no appreciable amounts of o- or m-tyrosine. Saliva samples from the same subjects after chewing betel quid to which 20 mg phenylalanine had been added contained o- and m-tyrosine at concentrations ranging from 1010 to 3000 nM and from 1110 to 3140 nM respectively. These levels were significantly higher (P < 0.005) than those of subjects who kept phenylalanine in the oral cavity without betel quid, which ranged from 14 to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine. These studies clearly demonstrate that the HO. radical is formed in the human oral cavity during betel quid chewing and is probably implicated in the genetic damage that has been observed in oral epithelial cells of chewers.

Topics: Areca; Calcium Compounds; Carbonates; Catechin; Cations, Divalent; Chromatography, High Pressure Liquid; Copper; Dimethyl Sulfoxide; Ethanol; Free Radical Scavengers; Humans; Iron; Isomerism; Mannitol; Mastication; Oxides; Phenylalanine; Plants, Medicinal; Reactive Oxygen Species; Reference Values; Root Canal Filling Materials; Saliva; Tyrosine

Topics: Animals; Chromatography, High Pressure Liquid; Male; Phenylalanine; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Tyrosine

Topics: Animals; Brain; In Vitro Techniques; Isomerism; Phenylalanine; Rats; Tyrosine