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Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The Km of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT’s and TAT’s biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.

Main conclusion Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for l-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. Abstract The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of l-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying l-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC–MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana. As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower (13)C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta. Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

Tyrosine aminotransferase (TAT, EC 2.6.1.5) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived pathway for syntheses of important secondary metabolites and compounds. Although plant TAT genes have been proposed to be important in response to abiotic stress, there is little information about TAT genes in woody perennial tree species, especially in economic fruit trees. Based on TAT domain searching, sequence homology screening and phylogenetic analysis, we identified four TATs in apple genome. Then, we carried out a detailed phylogenetic analysis of TAT genes from multi-species, focusing on apple (Malus domestica). The result showed that the TAT family comprises three major classes corresponding to genes from angiosperms, mammals, and bacteria. Angiosperm TAT genes could be further divided into six subclasses. Analysis of intron-exon structure revealed that the typical TAT gene contains six introns and seven exons, with exons of similar size at each exon location. Promoter analysis showed that the 5'-flanking region of apple MdTATs contain multiple cis-acting elements including those implicated in light, biotic stress, abiotic stress, and hormone response. MdTATs were expressed to various levels in all apple structures and organs evaluated, and showed distinct expression patterns under water deficit stress. Ectopic expression of MdTAT2 in Arabidopsis or over-expression of MdTAT2 in apple callus tissue conferred enhanced tolerance to drought and osmotic stress. Collectively, these results suggest a role for TAT genes in drought and osmotic stresses and provide valuable information for further research of TAT genes and their function in plants.

The current drugs for treating Leishmaniasis are toxic, non-economical and with the emergence of drug resistance makes the need for novel therapeutics urgent and necessary. In the current study, we report the identification of compounds TI 1-5 against tyrosine aminotransferase of L. donovani from a curated ZINC15 database containing 183,659 compounds. These flavonoid compounds had binding energies-8 kcal/mol and interacted with the active site residues S151, K286, C290, and P291. Assessment of physicochemical descriptors and ADMET properties established the drug likeliness of these compounds. The all-atom molecular dynamic simulations of the TAT-TI complexes exhibited stable geometrical properties and further trajectory analysis revealed the high-affinity interactions of TI 1, 3, 4, and 5 with the active site residues. DFT calculations reported the high electrophilic nature of TI 2 while other TI compounds demonstrated good kinetic stability and reactivity. From in vitro studies, TI 3 and TI 4 had the highest inhibition with Ki values of 0.9 ± 0.2 μM and 0.30 ± 0.1 μM, respectively. Taken together, the results from this study indicate the potentiality of TI 1, 3, 4, and 5 as anti-leishmanial leads, and these compounds can be exploited to manage the growing Leishmaniasis crisis in the world.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

The glucocorticoid status and activities of ALT, AST, and tyrosine aminotransferase in the liver are studied in rats with streptozotocin-induced diabetes mellitus in response to repeated intraperitoneal injections of mifepristone. Diabetic rats develop an increase of the blood corticosterone and liver aminotransferase levels in response to mifepristone. These results indicate that in diabetic animals the glucocorticoid hormones with high blood concentrations, increasing still more in response to mifepristone, overcome the receptor blockade, and realize their regulatory functions in hepatocytes. The effects of mifepristone on ALT activity are the most manifest. In normal rats, only ALT activity is increasing in response to mifepristone, while in rats with streptozotocin-induced diabetes mellitus, ALT activity increases more intensely than activities of tyrosine aminotransferase and AST.

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPβ) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.

Plants produce various L-tyrosine (Tyr)-derived compounds that are of pharmaceutical or nutritional importance to humans. Tyr aminotransferase (TAT) catalyzes the reversible transamination between Tyr and 4-hydroxyphenylpyruvate (HPP), the initial step in the biosynthesis of many Tyr-derived plant natural products. Herein reported is the biochemical characterization and subcellular localization of TAT enzymes from the model plant Arabidopsis thaliana. Phylogenetic analysis showed that Arabidopsis has at least two homologous TAT genes, At5g53970 (AtTAT1) and At5g36160 (AtTAT2). Their recombinant enzymes showed distinct biochemical properties: AtTAT1 had the highest activity towards Tyr, while AtTAT2 exhibited a broad substrate specificity for both amino and keto acid substrates. Also, AtTAT1 favored the direction of Tyr deamination to HPP, whereas AtTAT2 preferred transamination of HPP to Tyr. Subcellular localization analysis using GFP-fusion proteins and confocal microscopy showed that AtTAT1, AtTAT2, and HPP dioxygenase (HPPD), which catalyzes the subsequent step of TAT, are localized in the cytosol, unlike plastid-localized Tyr and tocopherol biosynthetic enzymes. Furthermore, subcellular fractionation indicated that, while HPPD activity is restricted to the cytosol, TAT activity is detected in both cytosolic and plastidic fractions of Arabidopsis leaf tissue, suggesting that an unknown aminotransferase(s) having TAT activity is also present in the plastids. Biochemical and cellular analyses of Arabidopsis TATs provide a fundamental basis for future in vivo studies and metabolic engineering for enhanced production of Tyr-derived phytochemicals in plants.

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine to p-hydroxyphenylpyruvate. Accumulation of tyrosine in the body due to a genetic mutation in the TAT gene causes tyrosomia type II in humans. The TAT gene is regarded as a model for studying steroid-inducible factors regulating a variety of biological functions of TAT. However, little is known of the effects of estrogen on the expression of the TAT gene in chickens. Therefore, in the present study, we identified expression of the avian TAT gene in various organs. The results showed the TAT was detected predominantly in the liver and reproductive organs including testis, oviduct, and ovary. Specifically, TAT mRNA was expressed abundantly in the glandular and luminal epithelia of the oviducts in response to endogenous and exogenous estrogens which also induce dramatic morphological changes in the oviduct of chickens. In addition, target microRNAs of TAT (miR-1460, miR-1626-3p, miR-1690-5p, and miR-7442-3p) were found to modulate expression of the TAT gene. Especially, miR-1690-5p influenced TAT gene transcription by binding directly to its 3'-UTR region. Moreover, the expression of TAT was abundant in glandular epithelia of cancerous but not normal ovaries from laying hens. Taken together, our findings suggest that TAT plays an important role in the cytodifferentiation of oviducts in response to estrogen and in the progression of ovarian cancer in chickens.

OBJECTIVEDespite an aggressive multimodal therapeutic regimen, glioblastoma (GBM) continues to portend a grave prognosis, which is driven in part by tumor heterogeneity at both the molecular and cellular levels. Accordingly, herein the authors sought to identify metabolic differences between GBM tumor core cells and edge cells and, in so doing, elucidate novel actionable therapeutic targets centered on tumor metabolism.METHODSComprehensive metabolic analyses were performed on 20 high-grade glioma (HGG) tissues and 30 glioma-initiating cell (GIC) sphere culture models. The results of the metabolic analyses were combined with the Ivy GBM data set. Differences in tumor metabolism between GBM tumor tissue derived from within the contrast-enhancing region (i.e., tumor core) and that from the peritumoral brain lesions (i.e., tumor edge) were sought and explored. Such changes were ultimately confirmed at the protein level via immunohistochemistry.RESULTSMetabolic heterogeneity in both HGG tumor tissues and GBM sphere culture models was identified, and analyses suggested that tyrosine metabolism may serve as a possible therapeutic target in GBM, particularly in the tumor core. Furthermore, activation of the enzyme tyrosine aminotransferase (TAT) within the tyrosine metabolic pathway influenced the noted therapeutic resistance of the GBM core.CONCLUSIONSSelective inhibition of the tyrosine metabolism pathway may prove highly beneficial as an adjuvant to multimodal GBM therapies.

Leishmania donovani tyrosine aminotransferase (LdTAT) is an essential enzyme that catalyzes the first step of amino acid catabolism. To understand LdTAT activity at different pH, molecular dynamics simulations were performed and trajectory and T-pad analysis pad were conducted. Fluorescence spectroscopy of LdTAT at various pH was measured to understand structural stability. UV studies on PLP were performed to determine the binding of the enzyme to cofactor PLP at different pH. The MD simulations showed that the structure of LdTAT was stable and no structural denaturation was observed at pH 2, 7 and 12. LdTAT exhibited the highest activity at pH -8 and fluorescent spectroscopy also corroborated by exhibiting the highest intensity at pH -8. Moreover, no structural denaturation was observed during the pH gradient. UV studies concluded that the aldimine bond forms only around neutral pH and redshift was observed on enzyme binding. From our observation, we hypothesize that the activity of LdTAT is a close interplay between the structure and charges of K286 and PLP. This study may provide significant insight into understanding parasitic enzymes like LdTAT during the life-cycle of Leishmania parasite. Knowledge of such enzyme mechanisms can pave the way for the design and delivery of enzyme-specific inhibitors.

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer.

Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent Km of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46–95% and RA were decreased by 36–91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77–2.6-fold with increased 1.3–1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn’t provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.

Trypanosoma cruzi, the etiological agent of Chagas disease, lacks genes that encode canonical branched-chain aminotransferases. However, early studies showed that when epimastigotes were grown in the presence of 14C1-DL-leucine, the label was incorporated into various intermediates. More recently, our studies provided evidence that T. cruzi epimastigotes display a single ATP-dependent and saturable transport system that enables epimastigotes to uptake branched-chain amino acids (BCAAs) from the culture media. To extend our knowledge of the first step of BCAA catabolism, the ability of this parasite′s non-canonical broad specificity aminotransferases, such as tyrosine aminotransferase (TAT) and aspartate aminotransferase (ASAT), to transaminate these amino acids was investigated. Indeed, our results show that TAT and ASAT utilize BCAAs as substrates; however, both enzymes differ in their catalytic competence in utilizing these amino donors. For instance, ASAT transaminates isoleucine nearly 10-fold more efficiently than does TAT. This unique characteristic of TAT and ASAT allows to explain how BCAAs can be oxidized in the absence of a BCAA transaminase in T. cruzi. This article is protected by copyright. All rights reserved.

Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.

none 5 no This research was supported by funds from the University of Bologna (RFO2014) to S.B.and from the University of Urbino (RFO 2014) to V.S.. Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01–5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress. open Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania

Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods.

MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat’s heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function.

Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism, especially in tyrosinemia type II, which is caused by deficiency of tyrosine aminotransferase and provokes eyes, skin, and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in this study, we investigated the in vivo and in vitro effects of tyrosine on some parameters of energy metabolism in cerebral cortex of 14-day-old Wistar rats. We observed that 2 mM tyrosine inhibited in vitro the pyruvate kinase (PK) activity and that this inhibition was prevented by 1 mM reduced glutathione with 30, 60, and 90 min of preincubation. Moreover, administration of tyrosine methyl ester (TME) (0.5 mg/g of body weight) decreased the activity of PK and this reduction was prevented by pre-treatment with creatine (Cr). On the other hand, tyrosine did not alter adenylate kinase (AK) activity in vitro, but administration of TME enhanced AK activity not prevented by Cr pre-treatment. Finally, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions and this diminution was prevented by Cr pre-treatment. The results suggest that tyrosine alters essential sulfhydryl groups necessary for CK and PK functions, possibly through oxidative stress. In case this also occurs in the patients, it is possible that energy metabolism alterations may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias.

BACKGROUND: The mode of action of the grass herbicides cinmethylin and 5-benzyloxymethyl-1,2-isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS: Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4-hydroxyphenylpyruvate (4-HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L-tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4-HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION: The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides. Copyright © 2011 Society of Chemical Industry

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [(3)H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GR(dim)) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.

A case of tyrosinosis due to lack of soluble tyrosine aminotransferase is described. The first clinical sign of this disorder may be bilateral keratopathy. Treatment is diet with restriction of phenylalanine and tyrosine. The disorder is rare and must be differentiated from other conditions of tyrosinosis.

Administration of short-chain fructooligosaccharide (scFOS) is known to lower serum triglyceride levels in rats fed a high-fat diet, but the molecular mechanisms remain unclear. This study aimed to identify marker genes for lipid-lowering effect of scFOS administration. The changes in hepatic gene expressions in rats fed scFOS were investigated using DNA microarray and quantitative RT-PCR analysis. The DNA microarray showed that phytanoyl-CoA 2-hydroxylase 2 (Phyh2), lipoprotein lipase (Lpl) and tyrosine aminotransferase (Tat) were significantly affected by scFOS administration (p < .05). Since Lpl is involved in lipid metabolism, the up-regulation of Lpl in the liver can be a potential marker of the lipid-lowering effect of scFOS.

Administration of cortisol to an animal induces tyrosine aminotransferase (TAT) in the liver. A similar effect was observed after stimulation of resident liver macrophages (Kupffer cells) by dextran sulfate. Actinomycin D completely blocks enzyme induction both by cortisol and dextran sulfate, whereas their combined effect gives an additive result. In primary culture of hepatocytes, dextran sulfate inhibits TAT activity, but conditioned macrophage medium reliably increases enzyme activity in hepatocytes. However, incubation of isolated macrophages in the presence of dextran sulfate and such medium transfer into hepatocyte culture results in even more pronounced increase in TAT activity. In a combined culture of hepatocytes and non-parenchymal liver cells, reproducing intercellular interactions in vitro, cortisol and non-parenchymal cells exhibit an additive effect on TAT activity. These results show that liver macrophages release a factor of unknown nature launching the mechanism of TAT induction independently of cortisol, a classic TAT inducer.

Three different forms of hereditary tyrosinemia type II, with some of the features described for the Richner-Hanhart's syndrome, occur in mink (Mustela vison Schreb.). These disorders are inherited as simple autosomal recessive characters. The main symptoms of the diseases are watery dull eyes, frequent urination, alteration of the hair/skin on the toes and a highly elevated level of tyrosine in blood and urine. An enzyme defect in hepatic tyrosine aminotransferase (EC 2.6.1.5) is considered as the reason of these three forms of the mink disease. Differences between these forms of hereditary tyrosinemia in mink now described lie among other things in the time of onset, duration and severity of the disese.

Various biochemical differences exist between mammalian tyrosine aminotransferase (TAT) and its analogue in Trypanosoma cruzi (TcTAT), the causative agent of Chagas disease. Moreover, TcTAT is over-expressed in strains of the parasite that are resistant to benznidazole (BZ), a drug currently used in chemotherapy. TAT has thus been indicated as a potential target for the development of new chemotherapeutic agents. In the present study, the TcTAT gene has been characterised in 14 BZ-resistant and susceptible strains and clones of T. cruzi. A unique transcript of 2.0 kb and similar levels of TcTAT mRNA were observed in all parasite populations. TcTAT gene is organized in a tandem multicopy array and is located on 8 chromosomal bands that vary from 785–2500 kb. No amplification of TcTAT was observed in the parasite genome. A 42 kDa protein expressed by TcTAT was present in all T. cruzi samples. The results suggest that TcTAT is not directly associated with the T. cruzi drug resistance phenotype. However, it may act as a general secondary compensatory mechanism or stress response factor rather than as a key component of the specific primary resistance mechanism in T. cruzi.

Here, we study a cycle of long-term starvation followed by refeeding in relation to the kinetics of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in rainbow trout (Oncorhynchus mykiss). We determine SerDH- and TyrAT- specific activity at different substrate concentrations in liver and white muscle of juvenile trout starved for 70 days and then refed for 6 hr, 32 hr, 4 days, and 9 days. SerDH showed a hyperbolic kinetic with a K(m) for L-serine of 77.07+/-8.78 mM in the liver of control trout. After 70 days of starvation, the SerDH activity at saturate substrate concentration rose 100% over control. No significant changes were found in the K(m) values of the enzyme. After refeeding, the SerDH activity declined to control values. TyrAT also showed a hyperbolic kinetic with a K(m) for L-tyrosine of 1.86+/-0.12 and 2.55+/-0.57 mM in liver and white muscle, respectively. In starved trout, TyrAT activity in liver and white muscle was about 64 and 267%, respectively, higher than control. After 9 days of refeeding, the control values recovered, although, at 6 hr of refeeding, hepatic TyrAT activity was higher than that for starvation. This work shows that SerDH and TyrAT are present in rainbow trout and that the two enzymes have regulatory functions in the catabolism of their respective amino acids in this species.

OBJECTIVE: The aims of the study were to evaluate whether growth hormone could be beneficial in a model of hypercatabolism induced by glucocorticoids and to examine its effects on ACTH, corticosterone and IGF-1 levels. The effects of growth hormone on the expression of both glucocorticoid receptor and tyrosine aminotransferase were also evaluated. METHODS: Fifty Wistar rats were divided into five groups and treated as follows: (A) daily subcutaneous injection of growth hormone (4.8 IU/kg/day) and oral placebo, (B) daily injection of placebo and oral dexamethasone (3 mg/kg/day), (C) daily injection of growth hormone and oral dexamethasone, (D) daily injection of placebo and oral placebo, and (E) no treatment. The animals were decapitated seven days after initiating treatment. RESULTS: Growth hormone did not modify the weight loss induced by dexamethasone. Glucocorticoid receptor expression was significantly lower in group A than in group E. An increase in tyrosine aminotransferase was observed in group C. CONCLUSION: Growth hormone did not exert any beneficial effect in this model of hypercatabolism. Growth hormone decreased glucocorticoid receptor expression. This fact could explain its beneficial effect when protein hypercatabolism is not the predominant phenomenon. Growth hormone induced the hyperexpression of tyrosine aminotransferase, thus suggesting an amplifying effect on the glucocorticoid action.

The effects of mercury (Hg) on basal and dexamethasone-induced tyrosine aminotransferase (TAT) activity in rat liver were studied. Comparison of TAT activity after in vitro and in vivo mercury application revealed the influence of the metal only when applied in vivo, suggesting that the effects are expressed at the level of TAT gene transcription. Intraperitoneal administration of mercury at 1, 2 or 3 mg Hg kg−1 b.w. 4 h before decapitation was shown to stimulate the basal activity of TAT. The most prominent increase was observed 4 h after the metal administration. When applied at 1 and 2 mg Hg kg−1 b.w. mercury was also shown to reduce partially the extent of the enzyme induction by dexamethasone, which was injected intraperitoneally at 5 mg kg−1 b.w. 5 h before death. The highest dose of mercury (3 mg Hg kg−1 b.w.) almost completely abolished the dexamethasone effect. The finding that mercury increases basal activity of the enzyme while decreasing its induction by dexamethasone suggests that stimulatory effects of this metal on TAT activity are probably mediated by factors other than glucocorticoids. Copyright © 2005 John Wiley & Sons, Ltd.

The genetic tyrosinemias are characterized by the accumulation of tyrosine in body fluids and tissues. The most severe form of tyrosinemia, Type I, is a devastating disorder of childhood that causes liver failure, painful neurologic crises, rickets, and hepatocarcinoma. This disorder is caused by a deficiency of fumarylacetoacetate hydrolase (FAH). If untreated, death typically occurs at less than 2 years of age, with some chronic forms allowing longer survival. It has a prevalence of about 1 in 100,000 newborns in the general population. Oculocutaneous tyrosinemia, Type II, is caused by a deficiency of tyrosine aminotransferase (TAT). It clinically presents with hyperkeratotic plaques on the hands and soles of the feet and photophobia due to deposition of tyrosine crystals within the cornea. Tyrosinemia Type III is an extremely rare disorder caused by a deficiency of 4-hydroxyphenylpyruvic dioxygenase. It has been associated with ataxia and mild mental retardation. These disorders are diagnosed by observing elevated tyrosine by plasma amino acid chromatography and characteristic tyrosine metabolites by urine organic acid analysis. In tyrosinemia Type I, methionine is also elevated, reflecting impaired hepatocellular function. Urine organic acids show elevated p-hydroxy-phenyl organic acids in each type of tyrosinemia, and the pathognomic succinylacetone in tyrosinemia Type I. Diagnosis can be confirmed by enzyme or molecular studies in tyrosinemia Type I. Therapy consists of a diet low in phenylalanine and tyrosine for each of the tyrosinemias and 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) for tyrosinemia Type I.

Tocopherols are presumed to be important antioxidants and scavengers of lipid radicals and reactive oxygen species in plants. Age is known to be a condition under which oxidative stress increases. In leaves of aging Arabidopsis thaliana plants, the content of alpha-tocopherol as well as of gamma-tocopherol increased significantly. The activity of tyrosine aminotransferase, which supplies the biosynthetic pathway with 4-hydroxyphenylpyruvate, was increased as well. On the other hand, coronatine, a phytotoxin mimicking octadecanoids and leading to symptoms of senescence, caused a moderate increase in alpha-tocopherol as well as some enhancement of gamma-tocopherol.

Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

Synthesis of L-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) can be induced by N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) in Reuber H35 cell cultures. Actinomycin D fails to block this induction which indicates a target for Bt2cAMP at a posttranscriptional level. We have determined the influence of Bt2cAMP on several translational events during the tyrosine aminotransferase synthesis with the following results. (1) The number of nascent tyrosine aminotransferase chains increased, whereas no effect was measured on the number of nascent total protein chains. (2) The rate of elongation along the tyrosine aminotransferase mRNA and total mRNA is not enhanced by Bt2cAMP. (3) The induced synthesis of tyrosine aminotransferase is more sensitive to the inhibition of elongation. We conclude from our results that Bt2cAMP induces the synthesis of tyrosine aminotransferase by an increase in the rate of initiation on the tyrosine aminotransferase mRNA.

The effects of rat-specific hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), mouse-specific hepatocarcinogen ortho-aminoazotoluene (OAT), non-species-specific hepatocarcinogen diethylnitrosamine (DENA), and non-carcinogenic 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA-binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen-susceptible and -resistant animals. Species-specific hepatocarcinogens 3'-MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non-carcinogenic 4'-MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA-binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species-specific effects of OAT and of 3'-MeDAB on HNF3 DNA-binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32-0.47 g/mL interval of ammonium sulfate concentration. In contrast, non-specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Previously we reported that intermittent intraperitoneal administration of ornithine decarboxylase-inducing factor (ODC factor), interleukin 1alpha (IL-1alpha), and tumor-necrosis factor-alpha (TNF-alpha) to normal mice induced biological changes in the hosts which included changes in the pattern of expression of pyruvate kinase (PK) isozymes in the liver and hypertrophy of the spleen. In the study reported here, we investigated the chronic and combined effects of these factors on hepatic enzymes using alzet microosmotic pumps implanted in the subcutis of the backs or abdominal cavities of mice. Continuous administration of ODC factor and recombinant human IL-1alpha (rhIL-1alpha) reduced the activity of L-type PK, which is a glycolysis-related enzyme in the liver, and induced the activity of M2-type PK, a known marker of liver dedifferentiation. Serine dehydratase (SDH) and tyrosine aminotransferase (TAT), enzymes associated with amino acid metabolism, were not significantly influenced at the examined concentration. The simultaneous continuous infusion of ODC factor and rhIL-1alpha or rhTNF-alpha caused alterations in the patterns of expression of PK isozyme activity profiles and reduced overall PK activity. SDH and TAT activities were also significantly induced. Moreover, mice treated with these combined factors displayed many other metabolic changes normally associated with cancer cachexia. These findings suggest that the tumor-derived ODC factor and cytokines such as IL-1alpha and TNF-alpha might function synergistically in the metabolic perturbations observed in Ehrlich ascites tumor bearers.

The results from a quantitative structure-property relationship (QSPR) model was integrated into a fifth-generation pharmacokinetic/pharmacodynamic (PK/PD) model of corticosteroid receptor/gene-mediated effects. The proposed model was developed using previously reported tyrosine aminotransferase (TAT) activity data following a 50 mg/kg intravenous dose of methylprednisolone in male adrenalectomized (ADX) rats. Induced TAT activity is a classical measure of corticosteroid genomic effects and the typical time course shows an initial lag-time, a slow rise to peak response, and a gradual return toward baseline values. The TAT activity profiles were subsequently predicted for two additional steroids (dexamethasone and hydrocortisone), which were confirmed experimentally. Two groups of male ADX Wistar rats (n = 18 each) were given either 0.1 mg/kg dexamethasone or 50 mg/kg hydrocortisone by penile vein injections. Plasma drug concentrations and liver TAT activity were measured at various time points. Baseline TAT activity was significantly lower in this study as compared to previous reports. Model simulations well captured the pharmacodynamic data once initial conditions were corrected for observed baseline values. Additional TAT profiles reported in the literature for prednisolone were also reasonably predicted using the final model. This study serves as a demonstration of how in vitro pharmacologic data and QSPR modeling results may be incorporated into existing mechanistic PK/PD models to anticipate the effects of other chemically related compounds.

3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) is a potent hepatocarcinogen in rats and a weak carcinogen in mice, whereas o-aminoazotoluene (OAT) is a potent hepatocarcinogen in mice but weak hepatocarcinogen in rats. They significantly suppress glucocorticoid induction of tyrosine aminotransferase (TAT) in the liver of sensitive animals and have minor effect on the induction of this enzyme in the liver of resistant animals (3'-MeDAB-treated mice and OAT-treated rats). The inhibitory effect of these carcinogens is realized at the level of gene transcription (decreased accumulation of TAT mRNA). This effect is mediated via reduction of DNA-binding activity of transcription factor HNF3 (without decrease of its content) without any involvement of the glucocorticoid receptor. It was shown that carcinogens influence DNA-binding activity of HNF3 via an unknown nuclear factor.

5-azacytidine (5-azaC) is known to induce cardiomyocyte differentiation. However, its function in hepatocyte differentiation is unclear. The present study investigated the in vitro capability of 5-azaC to promote maturation and differentiation of mouse embryonic hepatic progenitor cells, with the aim of developing an approach for improving hepatic differentiation. Mouse embryonic hepatic progenitor cells (HP14.5 cells) were treated with 5-azaC at concentrations from 0 to 20 μmol/l, in addition to hepatocyte induction culture medium. Hepatocyte induction medium induces HP14.5 cell differentiation. 5-azaC may enhance the albumin promotor-driven Gaussia luciferase (ALB-GLuc) activity in induced HP14.5 cells. In the present study 2 μmol/l was found to be the optimum concentration with which to achieve this. The expression of hepatocyte-associated factors was not significantly different between the group treated with 5-azaC alone and the control group. The mRNA levels of ALB; cytokeratin 18 (CK18); tyrosine aminotransferase (TAT); and cytochrome p450, family 1, member A1 (CYP1A1); in addition to the protein levels of ALB, CK18 and uridine diphosphate glucuronyltransferase 1A (UGT1A) in the induced group with 5-azaC, were higher than those in the induced group without 5-azaC, although no significant differences were detected in expression of the hepatic stem cell markers, DLK and α-fetoprotein, between the two groups. Treatment with 5-azaC alone did not affect glycogen synthesis or indocyanine green (ICG) metabolic function in HP14.5 cells, although it significantly increased ICG uptake and periodic acid-Schiff-positive cell numbers amongst HP14.5 cells. Therefore, the present study demonstrated that treatment with 5-azaC alone exerted no effects on the maturation and differentiation of HP14.5 cells. However, 5-azaC exhibited a synergistic effect on the terminal differentiation of induced hepatic progenitor cells in association with a hepatic induction medium.

5 p.-4 fig.-1 tab., The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding l-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 A ° resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis., This research was funded under Federal Contract No. HHSN272201200025C from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services. The project was also funded by grant AGL 2010-21806-C02-01 from the Spanish Ministry of Economy and Competitiveness and by contract No. 050204100014 from Fundación Ramón Areces. MAM thanks the National Research Council for grant 2012EST JAE Predoc.

PT/Y mice used for studies of the effects of mutagens are characterized by the absence of spontaneous tumors of the liver, but often develop these tumors in response to chronic oaminoazotoluene treatment. The level of glucocorticoid induction of adaptive hepatic enzyme tyrosine aminotransferase decreases by more than 70% 24 h after acute injection of o-aminoazotoluene to these animals. These mice can serve as a model for studies of the relationship between the effect of carcinogens on the regulation of activity of adaptive hepatic enzymes and their capacity to induce the development of liver tumors.

Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.

The aim of this study was to employ precursor-feeding strategy for the improved production of rosmarinic acid (RA) in Solenostemon scutellarioides in vitro. The cultures were fed with precursors, namely l-phenylalanine (Phe), l-tyrosine (Tyr) and cucumber juice (CJ), at different concentrations. Phe (100 mg L− 1) and Tyr (400 mg L− 1) caused ∼1.5- and 2.1-fold increase in RA accumulation within 48 h. CJ (50 mg L− 1) feeding displayed highest RA content (∼1.6-fold) in 72 h. In this study, we focused on the function of individual precursor on key enzymes involved in RA biosynthesis. The phenylalanine ammonia lyase activity was significantly upregulated after Phe (100 mg L− 1) feeding, while tyrosine aminotransferase and hydroxyphenylpyruvate reductase activities were improved with Tyr (400 mg L− 1) treatment. However, rosmarinic acid synthase activity was significantly enhanced by all three precursors. In synergy study, Phe (100 mg L− 1) + Tyr (400 mg L− 1) could enhance (∼3.1-fold) RA biosynthesis within 48 h.

We are studying the mechanisms of transcriptional activation by nuclear receptors and we focus our studies on the glucocorticoid regulation of the model tyrosine aminotransferase gene. Rather than using in vitro biochemical approaches, we determine the actual events occurring in the cells. Our experimental approaches include genomic footprinting, chromatin immunoprecipitation, in situ hybridization and transgenic mice. Our results show that the glucocorticoid receptor uses a dynamic multistep mechanism to recruit successively accessory DNA binding proteins that assist in the activation process. Chromatin is first remodelled, DNA is then demethylated, and the synthesis of an accessory factor is induced. Efficient transcription induction is finally achieved upon the formation of a 'stable' multiprotein complex interacting with the regulatory element. We discuss: the relative contribution of histone acetyltransferases and ATP-dependent remodelling machines to the chromatin remodelling event; the nature of the remodelled state; the contribution of regulated DNA demethylation to gene memory during development; the mechanisms of regulated DNA demethylation; the dynamics of protein recruitment at regulatory elements; the control of the frequency of transcription pulses and the control levels of the cell-type specificity of the glucocorticoid response.

The effects of mifepristone on activity of the adrenocortical system, expression of glucocorticoid receptor gene, and tyrosine aminotransferase activity in the liver were studied in rats with streptozotocin-induced diabetes. Administration of glucocorticoid receptor blocker mifepristone to rats without diabetes was followed by a 1.9-fold increase in serum corticosterone concentration and a 1.2-fold increase in tyrosine aminotransferase activity in the liver in comparison with the baseline values. In rats with streptozotocin-induced diabetes, mifepristone produced a less pronounced increase in the corticosterone concentration (by 1.5 times) and more drastic increase in enzyme activity (by 1.7 times). Mifepristone administration did not change the content of glucocorticoid receptor mRNA in the liver of rats without diabetes, but increase this parameter by 1.4 times in rats with streptozotocin-induced diabetes. The enhanced expression of glucocorticoid receptor gene in the liver of rats with streptozotocin-induced diabetes correlated with increased activity of tyrosine aminotransferase after mifepristone treatment.

Liver regeneration after two-thirds surgical partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). We have examined expression of the major liver-enriched transcription factors, cytochrome P450 (CYP) enzymes, and other markers of hepatocytic differentiation in SHPCs during the protracted period of liver regeneration after PH in retrorsine-exposed rats. Early-appearing SHPCs (at 3-7 days after PH) express mRNAs for all of the major liver-enriched transcription factors at varying levels compared to fully differentiated hepatocytes. In addition, SHPCs lack (or have significantly reduced) expression of mRNA for hepatocyte markers tyrosine aminotransferase and alpha-1 antitrypsin, but their expression levels of mRNA and/or protein for WT1 and alpha-fetoprotein (AFP) are increased. With the exception of AFP expression, SHPCs resembled fully differentiated hepatocytes by 14 days after PH. Expression of AFP was maintained by most SHPCs through 14 days after PH, gradually declined through 23 days after PH, and was essentially absent from SHPC progeny by 30 days after PH. Furthermore, early appearing SHPCs lack (or have reduced expression) of hepatic CYP proteins known to be induced in rat livers after retrorsine exposure. The resistance of SHPCs to the mitoinhibitory effects of retrorsine may be directly related to a lack of CYP enzymes required to metabolize retrorsine to its toxic derivatives. These results suggest that SHPCs represent a unique parenchymal (less differentiated) progenitor cell population of adult rodent liver that is phenotypically distinct from fully differentiated hepatocytes, biliary epithelial cells, and (ductular) oval cells.