Uptake Of Tyrosine Research Articles

Phenylalanine and Tyrosine Metabolism in Chronic Kidney Failure

In chronic kidney failure, there is impairment in the conversion of phenylalanine to tyrosine. As a result, tyrosine and the tyrosine/phenylalanine ratio are reduced in plasma and many tissues, and phenylalanine concentrations tend to be normal or slightly increased. Although animal studies indicate that the kidney is not a major contributor to the conversion of phenylalanine to tyrosine, human studies conducted in the postabsorptive state suggest that the kidney plays a major role in the uptake of phenylalanine and its hydroxylation and release as tyrosine. The human splanchnic bed in the postabsorptive state also displays net uptake of both phenylalanine and tyrosine and hydroxylation of substantial amounts of phenylalanine to form tyrosine. In chronic renal failure (CRF) patients, splanchnic uptake of tyrosine appears to be reduced in the postabsorptive state. After an amino acid meal, there is net release of phenylalanine from the splanchnic bed in normal subjects and to an even greater degree in CRF patients; tyrosine is released postprandially in both normal subjects and CRF patients. In the postabsorptive state, tyrosine release from the kidney is largely derived from the hydroxylation of phenylalanine. In CRF, the release of tyrosine from the kidney is reduced and this reduction may be marked with advanced CRF. These observations, as well as isotope studies indicating normal phenylalanine flux, reduced tyrosine flux and impaired conversion of phenylalanine to tyrosine in CRF patients, raise the possibility that tyrosine may be an essential amino acid in this condition. Further research will be necessary to answer this question. Oxidative stress, which often increases in CRF patients, may lead to increased formation of chlorotyrosine and nitrotyrosine in plasma proteins and of nitrotyrosine in the brain. Increased nitrotyrosine is also found in kidneys of patients with diabetic nephropathy or allograft nephropathy. Increased serum concentrations of oxidation products of phenylalanine have also been observed in patients with CRF. Impaired urinary excretion also may lead to accumulation of metabolic products of both phenylalanine and tyrosine in CRF. It is not known whether the elevated protein chlorotyrosine or nitrotyrosine or increased oxidative products of phenylalanine cause adverse metabolic or toxic effects in patients with CRF.

METABOLIC PHENOTYPING WITH POSITRON EMISSION TOMOGRAPHY FOR PREDICTION OF TUMOR RESPONSE TO THE EPIDERMAL GROWTH FACTOR RECEPTOR KINASE INHIBITOR ERLOTINIB (TARCEVA).

Objectives Recent studies have demonstrated that inhibition of epidermal growth factor receptor (EGFR) kinase activity rapidly reduces tumor glucose metabolism in sensitive cell lines. This suggests that imaging of tumor metabolism with positron emission tomography (PET) with the glucose analog [ 18 F]fluorodeoxyglucose (FDG) could be used clinically to monitor treatment with EGFR kinase inhibitors. In this study, I investigated whether EGFR kinase inhibition specifically affects glucose metabolism or whether it also affects lipid and amino acid metabolism. In doing so, I am attempting to determine the best PET tracers to identify malignancies responding to EGFR kinase inhibitors in the clinics. Methods The lung cancer cell lines H3255 and A549 served as models for tumors that are sensitive and resistant to EGFR kinase inhibition, respectively. Glucose, amino acid, and lipid metabolism were studied by measuring the uptake of radiolabeled analogs of glucose, tyrosine, and choline. Uptake of these tracers was assayed in vitro after incubating cells with increasing doses of the EGFR kinase inhibitor erlotinib and in vivo by PET imaging of tumor bearing mice before and after erlotinib treatment. In vitro blocking studies with specific reagents were performed to elucidate the mechanisms of tyrosine and choline uptake. Results In sensitive H3255 cells, erlotinib decreased glucose uptake 54 ± 2% within 4 hours, whereas tyrosine and choline uptake were unaffected. In resistant A549 cells, no reduction of tracer uptake was observed for any of the studied tracers. These in vitro findings were confirmed in H3255 xenografted mice, which demonstrated a selective reduction of glucose uptake by 50 ± 5% after two doses of erlotinib. In A549 xenografts, erlotinib had no measurable metabolic effects. In H3255 and A549 cells, 2-amino-2-norbornanecarboxylic acid, a specific inhibitor of l-type amino acid transport, reduced tyrosine uptake by 83 ± 1% and 75 ± 1%, and cold choline inhibited choline uptake with IC50s of 5 μM and 6 μM, respectively. Conclusion These observations suggest that efficacious erlotinib treatment specifically modulates glucose, but not amino acid or lipid metabolism, as EGFR kinase inhibition may not affect the l-type amino acid transport system or the high-affinity choline transport system. Thus, the glucose analog FDG is the most promising tracer for monitoring treatment with EGFR kinase inhibitors in the clinics.

Characterization of titanium dioxide nanoparticles imprinted for tyrosine by flow field-flow fractionation and spectrofluorimetric analysis

Films based on TiO 2 nanoparticles (NPs) have been successfully used as sensing elements in chemical sensors. TiO 2 colloidal suspensions can be obtained by spontaneous hydrolysis in acidic solutions of Ti(IV) compounds. The obtained TiO 2 NPs can be employed to build up nanostructured films. With the purpose of preparing TiO 2-based nanostructured, imprinted materials as sensing elements for piezoelectric sensors, we obtained TiO 2 NP dispersions by hydrolyzing potassium titanyl oxalate in the presence of a target analyte (tyrosine). Since morphological properties of the synthesized NPs are known to influence the nanostructured film characteristics, an analytical strategy to characterize such colloidal systems can combine a size-based separation method with spectroscopic analysis to correlate the particle size distribution (PSD) with the particle-target interaction properties able to determine the sensing efficiency. In this work, we present the characterization of colloidal tyrosine-TiO 2 NP systems by flow field-flow fractionation (FlFFF) with online, UV/Vis absorption detection and offline fluorescence analysis. FlFFF eliminates the possible contribution of free tyrosine to the absorption and fluorescence properties of the NPs. FlFFF also fractionates NPs on a size basis. Particle size distribution (PSD) profiles of the fractionated NPs are then obtained by conversion of the multi-wavelength UV/Vis fractograms. Size of the fractionated NPs is finally related to fluorescence properties of the collected NPs fractions. Good correlation between the fluorescence intensity, which is proportional to the tyrosine uptake, and the FlFFF-based, NP mass-size frequency distribution finally confirms the existence of tyrosine-TiO 2 NP interaction.

Tyrosine transport in fibroblasts from healthy volunteers and patients with schizophrenia

Aberrant tyrosine transport across the fibroblast membrane, as measured by lower V max and/or lower K m is a repeated finding in patients with schizophrenia. The aim of this study was to investigate the importance of two major transporters, the L- and A-systems and tyrosine transport in fibroblast cell lines from patients with schizophrenia and healthy volunteers. Fibroblast cell lines, n = 6 from healthy volunteers and n = 6 from patients with schizophrenia, were included in the study. Uptake of [14-C] l-tyrosine in fibroblasts was measured using the cluster tray method in absence and presence of inhibitors. The uptake of tyrosine by the L-system was evaluated with the inhibitor 2-aminobicyclo heptane-2-carboxylic acid (BCH) and the A-system with the inhibitor nonmetabolized methyl-aminoisobutyric acid (MeAIB). Using [14-C] MeAIB the functionality of system A isoform 2, ATA2, was tested. BCH inhibited the uptake of tyrosine with 90%, showing that tyrosine transport in fibroblasts is mainly transported by the L-system. Not more than 10% could be contributed by the A-system. Excess of MeAIB did not influence tyrosine kinetics. Moreover, MeAIB kinetics did not differ between the patients and the controls. In conclusion, aberrant tyrosine transport observed in patients with schizophrenia is probably linked to the one of the L-systems and does not seem to involve the ATA2 transporter.

A Family of Basic Amino Acid Transporters of the Vacuolar Membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

Momordica charantia Extracts Inhibit Uptake of Monosaccharide and Amino Acid across Rat Everted Gut Sacs in-Vitro

The inhibitory effects of Momordica charantia extracts were studied on the uptake of glucose and tyrosine across rat everted gut sacs in vitro. The aqueous extract of the plant was found to inhibit primarily the uptake of glucose in a dose-dependent manner. Uptake of tyrosine was affected at high substrate concentrations only. The extract was also found to decrease the absorptive capacity of fluid across the small intestine and sodium ions. It is hypothesized that the effects of Momordica could involve a washout of glucose from the blood stream.

Genotypic differences in the Chlamydia pneumoniae tyrP locus related to vascular tropism and pathogenicity.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes respiratory infections and has been associated with cardiovascular disease. We compared respiratory and cardiovascular isolates to find genetic differences associated with pathogenicity. A polymorphic region encoding a tyrosine/tryptophan permease was found to differ between disease isolates. Respiratory strains contained multiple copies of the tyrP gene, and vascular strains contained a single copy. Single-nucleotide polymorphism analysis revealed the duplication to be a phylogenetically old event. Gene amplification was associated with higher mRNA levels and higher uptake of the substrate tyrosine, indicating an amino-acid transport-related phenotype associated with the tyrP genotype. Vascular strains, despite their reduced ability to transport tyrosine, do not appear to have a reduced growth rate in vitro. We hypothesize that the important difference between strains of vascular and respiratory origin may lie in the increased tendency of vascular strains to elicit persistent infection that is triggered by amino-acid starvation.

Centrifugal and chromatographic analyses of tryptophan and tyrosine uptake by red blood cells and GLUT1 proteoliposomes with permeability estimates and observations on dihydrocytochalasin B

We analyzed transport into liposomes and proteoliposomes, separated the free and internalized radioactively labeled substrates by size-exclusion chromatography (SEC) and observed a net influx owing to nonfacilitated diffusion across the lipid bilayers during the separation. The permeabilities (10 −9 cm/s) of glucose transporter (GLUT1) proteoliposomes were estimated to be 4.6, 1.0, 1.4 and 2.1 for d-glucose, l-glucose, l-Tyr and l-Trp, respectively; 15, 3.3, 5.1 and 2.1 times higher than the corresponding permeabilities of liposomes. These values indicated that GLUT1 did not transport Tyr or Trp, or transported Tyr, and only Tyr, slowly. This interpretation was supported by further analyses. Dihydrocytochalasin B inhibited the transport of Tyr and, partially, Trp into human red blood cells (centrifugal analyses). It did not inhibit Tyr and Trp influx into GLUT1 proteoliposomes, but partitioned strongly into the bilayers and seemed to make them fragile. The GLUT1 inhibitor cytochalasin B and the GLUT1 substrate 2-deoxy- d-glucose did not inhibit Tyr transport into the cells. Upon immobilized biomembrane affinity chromatography, Trp decreased the cytochalasin B retardation by GLUT1 only at levels far above the physiological Trp concentration. Ethanol (commonly added to aqueous solutions for enhancing a compound's solubility) halved the retardation at 4% (v/v) concentration. Drastic modification of the SEC method is required to allow permeability measurements with nonlabeled and highly permeable substrates.

Thyroid Hormone Transport by the Heterodimeric Human System L Amino Acid Transporter

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01–10μ m [125I]T4,[ 125I]T3,[ 125I]rT3, or[ 125I]3,3′-diiodothyronine or with 10–100μ m [3H]arginine, [3H]leucine,[ 3H]phenylalanine, [3H]tyrosine, or[ 3H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 μm) and of 3,3′-diiodothyronine > rT3 ∼ T3 > T4 (10 nm), which in all cases was Na+ independent. Saturation analysis provided apparent Michaelis constant (Km) values of 7.9 μm for T4, 0.8 μm for T3, 12.5μ m for rT3, 7.9 μm for 3,3′-diiodothyronine, 46 μm for leucine, and 19μ m for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 μm) was inhibited by the different iodothyronines (10 μm), in particular T3. Vice versa, uptake of 0.1 μm T3 was almost completely blocked by coincubation with 100μ m leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na+-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.

Thyroid hormone transport by the heterodimeric human system L amino acid transporter.

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine, [(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.

Effects of tumor necrosis factor alpha on taurine uptake in cultured rat astrocytes

Taurine is known to play a major role in volume regulation in astrocytic swelling associated with stroke and brain trauma. Apart from brain edema, the severity of brain injury is related to the levels of inflammatory cytokines such as tumor necrosis factor α (TNFα). TNFα had been shown to be closely associated with brain edema formation since the neutralization of TNFα reduced brain edema. Considering taurine has osmoregulatory functions in astrocytes, experiments were performed to study the effects of TNFα on taurine uptake in cultured astrocytes. Astrocytes exposed to 20 ng/ml of TNFα for 48 h showed a 91% increase in taurine uptake and significant increase was observed after 24 h exposure. This cytokine caused neither significant changes in cell volume nor taurine release. The increased in taurine uptake induced by TNFα was unlikely resulted from the modification of Na + movement because TNFα decreased tyrosine uptake, Na +-dependent transport system. In contrast to TNFα, interferon-γ (IFNγ) did not significantly affect taurine uptake. Taken together, our results did not support a suggestion that TNFα affects cell volume regulation via modulating taurine uptake in astrocytes. Increasing lines of evidence have demonstrated that taurine has anti-inflammatory and anti-oxidative effects, these findings therefore suggested that the increase in taurine uptake might be an adaptive response or a tool for astrocytes against oxidative stress.

Lead and Ethanol Coexposure: Implications on the Dopaminergic System and Associated Behavioral Functions

The present investigation involves ethanol's effects on the lead-induced alterations in the dopaminergic system. Ethanol, at a dose of 3 g/kg body weight for 8 weeks, resulted in a marked increase in the accumulation of lead in the blood and brain of animals receiving 50 mg lead/kg body weight. Levels of dopamine were found to decrease significantly, and were accompanied with increased norepinephrine levels in lead and ethanol coexposed animals. Uptake of tyrosine as well as the activities of tyrosine hydroxylase and monoamine oxidase were seen to increase significantly in lead as well as ethanol-treated animals, and these were increased to a greater extent when animals were administered lead and ethanol simultaneously. Dopamine receptor binding studies revealed a significant elevation in the number of binding sites in lead and ethanol-coexposed animals. The altered dopaminergic functions were reflected by the neurobehavioral deficits in terms of motor incoordination, aggressiveness, and hyperactivity of animals exposed to lead, the effect being more pronounced in lead- and ethanol-coexposed animals. In brief, results of this study suggests that ethanol potentiates lead-induced cellular damage at the neurochemical and neurobehavioral level.

Downregulation of Tyrosinase Activity in Human Melanocyte Cell Cultures by Yohimbine

Treatment of human melanocyte cell cultures with the alpha-2 adrenergic receptor antagonist yohimbine results in a marked down-regulation of tyrosinase activity. A 30% decrease occurs within 12 h of exposure of cells to yohimbine (100 microM), and by 48 h tyrosinase activity in treated melanocytes is less than a fifth that of control cultures. The inhibition is dose dependent and occurs in human melanocytes derived from either black or white skin types, and also in mouse melanoma cells. The yohimbine-induced decrease in tyrosinase activity is reversible, with enzyme levels returning to 90% of control values 48 h after removal of drug. Although tyrosinase activity is markedly suppressed by yohimbine, the compound has no effect on cell proliferation, cellular translation, or DNA synthesis. Treatment of melanocyte cultures with yohimbine blocks the increase in tyrosinase activity by either 3-isobutyl-1-methylxanthine, dibutyryl cAMP, or forskolin. Results of cAMP immunoassays, show that intracellular levels of the cyclic nucleotide are unaffected in cells treated with yohimbine. Tyrosinase inhibition by yohimbine does not involve a decrease in substrate availability since tyrosine uptake studies show that yohimbine has no effect on the amount of tyrosine entering the cell. Incubation of a melanosome-enriched fraction with yohimbine does not cause a lowering of tyrosinase activity, suggesting that an intact cell is required for yohimbine action. In addition, tyrosinase extracts show no reduction in activity when incubated directly with yohimbine, indicating that the drug does not act as a direct inhibitor of the enzyme. Finally, results of western immunoblotting show that yohimbine does not significantly lower the amount of tyrosinase protein in human melanocytes. These findings suggest that yohimbine acts through an as yet unidentified signaling pathway to lower the catalytic activity of pre-existing tyrosinase molecules present in melanocytes.

Effect of UV irradiation on cell surface protease activity and amino acid uptake

The surface of most cells includes a coterie of resident proteins which act as receptors for a wide variety of ligands and other proteins which are potentially bioactive on cell–cell contact (juxtacrine effects), or else are released by enzyme activity to influence cell behaviour by autocrine or paracrine mechanisms. We previously found that UVC irradiation stimulates the release of TGFα from its membrane-bound preprocursor form whereby it acts as a stimulus to rapid, reparative cell multiplication; clearly this runs the risk hastening mitosis before UV-induced DNA damage is fully corrected, which in turn may increase the likelihood of residual lesions persisting and hence of new mutations being generated. We found that sublethal UVC irradiation (10 J m −2) of HeLa cell cultures also resulted in activation of ecto-aminopeptidase and ecto-endopeptidases which were maximal 16 and 20–24 h after irradiation, respectively. Both of these classes of protease were shown to be metalloproteases using a nonapeptide substrate (called P9) which is cognate to the N-terminal cleavage site of preproTGFα except for a reporter 125 I -tyrosine [Piva et al., J. Cell. Biochem. 64 (1997) 353–368]. We now show that the N-terminal tyrosine cleaved from P9 by cell surface aminopeptidase activity, was found to be taken up by the cell resulting in its 10–25-fold concentration intracellularly, some two- to threefold higher than from a reservoir source, and may represent a novel salvage pathway for recovery of essential amino acids. Aminopeptidase activity was found to be both temperature- and FBS-dependent but was not reliant on ATP for its activity. Tyrosine transport across the cell membrane was also temperature and FBS-dependent but required ATP for maximal activity. UVC irradiation enhanced aminopeptidase activity but not tyrosine uptake by the cultures. The fraction of HeLa cells undergoing apoptosis increased in those cultures which were exposed to higher doses of UVC. The levels of ecto-aminopeptidase and ecto-endopeptidase activity in apoptotic cells were elevated compared to viable cells receiving the same dose of UVC. These results suggest that increased levels of cell surface protease activity in apoptotic cells would increase the amounts of free amino acids and growth factors in the extracellular medium and hence stimulate the proliferation of surrounding cells to replace those killed by UV irradiation.

Tyrosine Transport into Melanosomes Is Increased Following Stimulation of Melanocyte Differentiation

A variety of physiological factors can stimulate differentiation of melanocytes to increase pigmentation, and critical to this process is the transport of the melanogenic substrate (tyrosine) into melanosomes. In this study, we examined whether stimulation of melanogenesis affects melanosomal tyrosine transport. Tyrosine uptake increased almost 2-fold in melanosomes derived from melanocytes treated with melanocyte-stimulating hormone (MSH), which acts to increase intracellular cAMP levels, resulting in the up-regulation of many genes involved in melanogenesis. Stimulation of melanoma cells with dibutyryl cAMP increased melanosomal tyrosine transport 2- to 3-fold after 24 to 48 hrs, with peak levels occurring after 3 to 5 days of treatment, suggesting thatde novogene expression may be required. The cAMP-induced increase in melanosomal tyrosine transport could be effectively competed with phenylalanine or tryptophan, but not with dopamine or proline, suggesting either that a pool of transporters with greater tyrosine transporting ability pre-exists, or that a greater number of tyrosine transporters reside within the melanosomal membrane. These results illustrate a rare example of hormonal plasma membrane stimulation which transduces a signal for increased vesicular transport of an amino acid.

Tyrosine transporter in larval Leptinotarsa decemlineata midgut brush border membrane

An ion-dependent tyrosine transporter from Leptinotarsa decemlineata midgut was further characterized using brush border membrane vesicles. Tyrosine transport is not stimulated by Li +, Rb +, Cs +, Ca 2+, or Mg 2+. K + paired with SCN − stimulates tyrosine uptake, but K + paired with less permeant PO 4 2− or gluconate anions does not stimulate tyrosine uptake. KSCN stimulates initial tyrosine uptake in a concentration-dependent manner, reaching a maximum at 20 mM. Tyrosine transport is slower when moving against a positive electrical gradient than against a negative or neutral electrical gradient. In the presence of an inward K + gradient, tyrosine is not concentrated in BBMV above the equilibrium concentration. Tyrosine transport is highest at pH 6.4 and 7.4, and significantly lower at pH 9.4. This ion-dependent tyrosine transporter is insensitive to the amino acid analogs methylamino-isobutyric acid and 2-aminobicyclo-[2,2,11-heptane-2-carboxylic acid. None of eight plant lectins tested inhibited initial tyrosine uptake. The Bacillus thuringiensis Cry3A ∂-endotoxin, active against L. decemlineata in vivo, did not affect initial tyrosine uptake rate.

Schistosoma mansoni: Effect of recombinant tumor necrosis factor α on fecundity and [ 14C]-tyrosine uptake in females maintained in vitro

It has been reported that recombinant tumor necrosis factor α (rTNFα) stimulates egg-laying in Schistosoma mansoni females. Because tyrosine requirement is increased in females undergoing sexual maturation in preparation for oögenesis and tyrosine is a major component of eggshell protein, we wanted to determine whether females treated with rTNFα would also incorporate more tyrosine. Adult females were first treated with 10, 20 or 40 ng/ml rTNFα for 1, 3 or 6 hr in RPMI 1640 containing 10% fetal calf serum (FCS). Another two groups of females were each exposed either to males or to male excretory-secretory (ES) products for 1 hr. They were then exposed to 20 μCi/ml −1 [ 14C]-tyrosine for 1 hr in RPMI 1640 containing 10% FCS. All females were incubated individually unless indicated otherwise. Females incorporated more tyrosine after exposure to males or their ES products. They incorporated significantly more tyrosine when treated with rTNFα for 1 hr; the increased uptake correlated with increasing amounts of rTNFα used. Although after a 3-hr treatment with 10 ng/ml rTNFα females incorporated slightly more tyrosine than controls, increasing amounts of rTNFα had an adverse effect. Females treated with rTNFα for 6 hr incorporated less tyrosine than controls and those treated for 1 hr. SDS-PAGE and fluorography did not reveal any differences in polypeptide profiles of untreated and rTNFα-treated females. These unexpected results led us to study the effect of rTNFα on fecundity in females. Contrary to the published report, we observed a sharp decline in egg-laying in females when exposed to increasing concentrations of rTNFα in vitro.

Effects of amino acids on synthesis and degradation of skeletal muscle proteins in humans.

Synthesis and degradation of globular and myofibrillar proteins across arm and leg muscles were examined during stepwise increased intravenous infusion of amino acids (0.1, 0.2, 0.4, and 0.8 g N.kg-1.day-1) to healthy volunteers. Protein dynamics were measured by a primed constant infusion of L-[ring-2H5]phenylalanine and the release of 3-methylhistidine from skeletal muscles. Arterial concentrations and flux of glucose, lactate, and free fatty acids were unchanged despite increasing concentrations of plasma amino acids from 2.6 to 5.7 mM. Plasma insulin, insulin-like growth factor I (IGF-I), and plasma concentrations of IGF-I-binding proteins-1 and -3 remained at fasting levels throughout the investigation. Amino acid infusion caused a significant uptake of the majority of amino acids across arm and leg tissues, except tyrosine, tryptophan, and cysteine, probably due to low concentrations of these amino acids in the formulation. The balance of globular proteins improved significantly (P < 0.01) due to stimulation of synthesis and attenuation of degradation across arm and leg tissues, despite insignificant uptake of tyrosine, tryptophan, and cysteine. Degradation of myofibrillar proteins was uninfluenced by provision of amino acids. The results demonstrate that neither insulin nor circulating IGF-I explained improved protein balance in skeletal muscles after elevation of plasma amino acids. Rather, some amino acids in themselves trigger cellular reactions that initiate peptide formation. Limited availability of some extracellular amino acids was overcome by increased reutilization of the intracellular amino acid.

Tyrosine uptake and regional brain monoamine metabolites in a rat model resembling congenital hyperammonemia.

Hyperammonemia found in congenital disorders has a toxic effect on the central nervous system. Disturbances of brain neurotransmitter metabolism have been proposed, such as an increased transport of tryptophan into the brain and an increased flux through the serotonin pathway. Results concerning the catecholamine pathway are, however, contradictory. We therefore studied whether hyperammonermia increases brain uptake of the neurotransmitter precursor amino acid tyrosine and whether these changes affect the concentration of neurotransmitters and their metabolites in different brain areas (frontal cortex, caudatus-putamen, thalamus, hypothalamus, hippocampus/substantia nigra, brainstem) of rats made hyperammonemic with urease. The brain uptake of tyrosine was measured in the forebrain, brainstem, and cerebellum. The brain areas were analyzed for dopamine, 3,4-hydroxyphenylacetic acid; homovanillic acid, norepinephrine, and vanillylmandelic acid. The brain uptake index of tyrosine was increased in the forebrain and brainstem of the hyperammonemic rats with concomitantly elevated concentrations in the forebrain of tyrosine, phenylalanine, and tryptophan. The homovanillic acid content was significantly increased in the hypothalamus, hippocampus/substantia nigra and brainstem. The concentrations of norepinephrine, dopamine, and 3, 4-hydroxyphenylacetic acid were not significantly changed. Vanillylmandellic acid was decreased in the caudatus-putamen, thalamus, and hypothalamus. The data indicate an undisturbed neurotransmitter synthesis and, taken with the augmented tyrosine uptake at the blood-brain barrier, an increased flux through the dopamine pathway. These changes observed in the hyperammonemic animal model could contribute to the understanding of the pathogenic mechanisms and offer an explanation for the neuropsychiatric disturbances observed in children with congenital hyperammonemia.

Neurotransmitter positron emission tomographic-studies in adults with phenylketonuria, a pilot study

Patients with phenylketonuria (PKU) may suffer from cognitive and neurological deficits which are related to reduced intracerebral concentrations of catecholamines. The function of phenylalanine (Phe) as an inhibitor of the uptake of the precursor amino acid tyrosine (Tyr) through the blood-brain barrier as well as an inhibitor of the expression of dopamine receptors in the brain is under investigation. Positron emission tomography (PET) is a method for quantitatively determining biochemical and physiological processes in vivo. In the current pilot study, L-[1-11C]-Tyr and 18F-fluoro-ethyl-spiperone (FESP) have been used. The metabolic pathway of carboxylic labelled Tyr is mainly incorporation into protein. From the measured tissue and plasma activity as a function of time in combination with a compartimental model the Protein Synthesis Rate (PSR) for Tyr can be calculated. FESP is a ligand which binds irreversibly to the dopamine D2-receptor and has also a low non specific binding, although affinity to the serotonin receptor has been described. The ratio of FESP concentration in striatum and in cerebellum is a measure of the receptor status in vivo. In patients with plasma Phe levels above the maximum therapeutic concentration (> 700 mumol/l) the PSR for Tyr was decreased as compared to controls and patients with plasma Phe concentrations within the therapeutic range, indicating a decreased availability of Tyr for neurotransmitter synthesis, and hence explaining the reduced cerebral concentration of catecholamines.