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Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up‑regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-β-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up‑regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.

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.

Background In 2012, Venet et al. proposed that at least in the case of breast cancer, most published signatures are not significantly more associated with outcome than randomly generated signatures. They suggested that nominal p-value is not a good estimator to show the significance of a signature. Therefore, one can reasonably postulate that some information might be present in such significant random signatures. Methods In this research, first we show that, using an empirical p-value, these published signatures are more significant than their nominal p-values. In other words, the proposed empirical p-value can be considered as a complimentary criterion for nominal p-value to distinguish random signatures from significant ones. Secondly, we develop a novel computational method to extract information that are embedded within significant random signatures. In our method, a score is assigned to each gene based on the number of times it appears in significant random signatures. Then, these scores are diffused through a protein-protein interaction network and a permutation procedure is used to determine the genes with significant scores. The genes with significant scores are considered as the set of significant genes. Results First, we applied our method on the breast cancer dataset NKI to achieve a set of significant genes in breast cancer considering significant random signatures. Secondly, prognostic performance of the computed set of significant genes is evaluated using DMFS and RFS datasets. We have observed that the top ranked genes from this set can successfully separate patients with poor prognosis from those with good prognosis. Finally, we investigated the expression pattern of TAT, the first gene reported in our set, in malignant breast cancer vs. adjacent normal tissue and mammospheres. Conclusion Applying the method, we found a set of significant genes in breast cancer, including TAT, a gene that has never been reported as an important gene in breast cancer. Our results show that the expression of TAT is repressed in tumors suggesting that this gene could act as a tumor suppressor in breast cancer and could be used as a new biomarker.

Endophyte is a factor that affects the physiology and metabolism of plant. However, limited information is available on the mechanism of interaction between endophyte and plant. To investigate the effects of endophytic fungus ZPRs-R11, that is, Trimmatostroma sp., on salidroside and tyrosol accumulations in Rhodiola crenulata, signal transduction, enzyme gene expression, and metabolic pathway were investigated. Results showed that hydrogen peroxide (H2O2), nitric oxide (NO), and salicylic acid (SA) involved in fungus-induced salidroside and tyrosol accumulations. NO acted as an upstream signal of H2O2 and SA. No up- or down-stream relationship was observed, but mutual coordination existed between H2O2 and SA. Rate-limiting enzyme genes with the maximum expression activities were UDP-glucosyltransferase, tyrosine decarboxylase (TYDC), monoamine oxidase, phenylalanine ammonialyase (PAL), and cinnamic-4-hydroxylase sequentially. Nevertheless, the genes of tyrosine transaminase and pyruvate decarboxylase only indicated slightly higher activities than those in control. Thus, TYDC and PAL branches were the preferential pathways in ZPRs-R11-induced salidroside and tyrosol accumulation. Trimmatostroma sp. was a potential fungus for promoting salidroside and tyrosol accumulations. The present data also provided scientific basis for understanding complex interaction between endophytic fungus and R. crenulata.

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.

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.

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.

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.

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.

After repeated administration of cocaine at intervals, sensitization phenomena can be observed, so that its behavioural effects are enhanced. Since this phenomenon is long-lasting, it was of interest to study which persistent alterations in the activity of dopaminergic neurones or of endogenous opioid systems downstream of dopaminergic synapses in the basal ganglia are involved in the sensitization. Cocaine (10 mg/kg i.p.) was administered to rats on days 1, 3, 5 and 7 and saline on days 2, 4 and 6 ("repeated cocaine"), or saline was injected on days 1-6 and cocaine on day 7 ("acute cocaine"), or saline was injected on days 1-7 ("saline group"). The "repeated cocaine" schedule led to a significant sensitization to the locomotor activation produced by cocaine on day 7 or on day 17, 10 days after the end of sensitization protocol. Microdialysis in the nucleus accumbens which was performed after administration of cocaine (10 mg/kg i.p.) on day 7, or after an administration of the same dose 10 days after the last administration of cocaine, respectively, revealed significant acute increases of extracellular dopamine to about 200% of basal values. These increases were similar in "acute cocaine" and in "repeated cocaine" animals both after 7 days and after 17 days. For in situ hybridization studies, rats were sacrificed on day 7, 4.5 h after the last cocaine or saline administration. The mRNA for tyrosine hydroxylase (TH) in substantia nigra + ventral tegmental area was significantly elevated to about 140% of saline controls both in the "repeated cocaine" and the "acute cocaine" group as compared with the "saline group". In contrast, there were no differences between the three groups in the mRNAs of preprodynorphin or preproenkephalin levels measured in the nucleus accumbens (core and shell). These results suggest that sensitization phenomena to cocaine are not necessarily connected with alterations in the dopaminergic activity in the mesolimbic system or in the transcription of precursors of endogenous opioid peptides which are located downstream of the dopaminergic synapses.

Hypoglycemic coma caused by insulin injection to rats with alloxan-induced diabetes was accompanied by an increase in the concentrations of urea and uric acid and decrease in the content of free amino acids in blood plasma. Activities of glutamate dehydrogenase, AMP deaminase, glutaminase, ALT, and AST in the liver of experimental animals increased.

To study the role of cell shape in control of hepatocyte function, we have developed a system that can quantitatively control the spreading of cultured rat hepatocytes using poly[2-hydroxyethyl methacrylate]. When hepatocytes were cultured in a dish coated with high concentration of poly[2-hydroxyethyl methacrylate] solution, formation of stress fibers were suppressed and they continued to have a compact shape. In the compact-shaped hepatocytes, the ability to induce tyrosine aminotransferase with dexamethasone remained high for longer periods of time, as compared to the hepatocytes that spread following culture in the polystyrene dish. Conversely, the hepatocytes showed more active DNA synthesis when they assumed a flat shape as a result of spreading. When the hepatocytes that had spread following long-term culture in the polystyrene dishes were treated with cytochalasin to induce depolymerization of F-actin, the ability of the cells to induce tyrosine aminotransferase upon stimulation with dexamethasone improved markedly. This effect was not altered by treatment with actinomycin D but was completely suppressed by cycloheximide, suggesting that microfilaments are involved in the post-transcriptional process of tyrosine aminotransferase induction. Thus, there is a possibility that F-actin rather than cell shape might regulate cellular function in primary cultured hepatocytes.

Suckling mice were more sensitive to the hepatocarcinogenic effect of various carcinogens compared to adult animals. After treatment with o-aminoazotoluene and diethylnitrosamine HNF3-DNA-binding capacity and glucocorticoid-induced liver tyrosine aminotransferase activity in suckling mice decreased more significantly than in adult animals.

A third-generation pharmacokinetic/pharmacodynamic model was proposed for receptor/gene-mediated corticosteroid effects. The roles of the messenger RNA (mRNA) for the glucocorticoid receptor (GR) in hepatic GR down-regulation and the mRNA for hepatic tyrosine aminotransferase (TAT) induction by methylprednisolone (MPL) were examined. Male adrenalectomized Wistar rats received 50 mg/kg MPL iv. Blood and liver samples were collected at various time points for a period of 18 hr. Plasma concentrations of MPL, free hepatic cytosolic GR densities, GR mRNA, TAT mRNA, and TAT activities in liver were determined. Plasma MPL profile was biexponential with a terminal t1/2 of 0.57 hr. Free hepatic GR density rapidly disappeared from cytoplasm after the MPL dose and then slowly returned to about 60% of starting level after 16 hr. Meanwhile, GR mRNA level fell to 45% of baseline within 2 hr postdosing, and remained at that level for at least 18 hr. The GR down-regulation of GR mRNA and protein turnover rate were modeled. The TAT mRNA began to increase at about 2 hr, reached a maximum at about 5 hr, and declined to baseline by 14 hr. TAT induction followed a similar pattern, except the induction was delayed about 0.5 hr. Pharmacodynamic parameters were obtained by fitting seven differential equations in a piecewise fashion. The cascade of corticosteroid steps were modeled by a series of inductions for steroid-receptor-DNA complex, two intermediate transit compartments, TAT mRNA, and TAT activity. Results indicate that GR mRNA and TAT mRNA are major controlling factors for the receptor/gene-mediated effects of corticosteroids.

A 25-year-old woman with tyrosinaemia type II was treated from the 5th week of pregnancy with a protein-restricted diet supplemented with a tyrosine/phenylalanine-free amino acid mixture. Tyrosine concentrations were maintained in the range 100-200 mumol/l by restricting natural protein intake to 0.16 g/kg per 24 h in early pregnancy, with increases up to 0.38 g/kg per 24 h in the last trimester. This treatment maintained plasma phenylalanine concentrations in the range 20-40 mumol/l. Maternal weight gain and fetal growth were normal, and the mother remained asymptomatic throughout the pregnancy. A normal infant was born at term with length, weight and head circumference between the 25-50th per centiles.

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.

Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey, Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4 X 10(9) cells with viabilities of 90.8 +/- 5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5'-nucleotide phosphodiesterase remained unchanged. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of alpha-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness, demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.

Our receptor/gene-mediated model of corticosteroid action was tested and extended by examining the pharmacokinetics/dynamics of multiple low doses vs. a single higher dose of intravenously administered prednisolone in adrenalectomized male Wistar rats. Low-dose rats received 3 bolus doses (5 mg/kg) of prednisolone at 0, 0.5 and 1.0 hr. High-dose animals were given a single 25 mg/kg dose of prednisolone. Both regimens were expected to produce equivalent net responses based on model predictions. Control rats were not dosed. The profiles of free hepatic cytosolic glucocorticoid receptors and the hepatic tyrosine aminotransferase (TAT) enzyme were examined. Plasma prednisolone concentrations showed bi-exponential decline for both doses using pooled animal data. Clearance of total plasma prednisolone was 4.16 and 3.21 L/hr per kg in low- and high-dose groups. Volume of distribution at steady state (approximately 1.50 L/kg) and central volume (approximately 0.6 L/kg) were similar for both groups. Receptor levels from 5-16 hr stabilized at 64% of the 0-hr control value. Receptor and TAT profiles were essentially superimposable for both dosing groups. Our previous model was used to simultaneously describe prednisolone plasma concentrations, hepatic receptors, and TAT activity. The ability of total plasma prednisolone (Cp), corticosteroid binding globulin (CBG)-free plasma prednisolone (CCBG), and free plasma prednisolone (CF) to describe the kinetics/dynamics were examined. The CF values produced optimum fitting of all receptor data. The similarity of the two dosing groups supports the view that appropriately timed doses of a steroid can be used in an optimally efficacious manner by first filling all receptor sites and then replacing steroid as receptors are expected to recycle from nuclear/DNA binding sites as the steroid is eliminated.

The zonal distribution of phosphoenolpyruvate carboxykinase (PCK) and tyrosine aminotransferase (TAT) mRNA in liver was studied by in situ hybridization with radiolabelled cRNA probes and the abundance of PCK and TAT mRNA was quantified by Northern blot analysis of total RNA with biotinylated cRNA probes. Livers were taken from rats during a normal 12 h day/night rhythm, when they had access to food only during the dark period from 7 pm to 7 am, or during refeeding, when they had access to food after having been starved for 60 h. 1. Daily feeding rhythm: High levels of PCK mRNA were distributed mainly in the periportal and intermediate zone during the fasting period at noon and 6 pm. Feeding caused a rapid decrease in PCK mRNA level and a restriction of PCK mRNA localization to the periportal area within the first 2 h. No further alterations were observed during the following hours of the feeding period. TAT mRNA was distributed also in the periportal and intermediate zone during the fasting period. Feeding first reduced the mRNA level without changing the distribution pattern. Then towards the end of the feeding period TAt mRNA increased again to half-maximal levels and became restricted mainly to the periportal area. 2. Starvation-refeeding cycle: High amounts of PCK mRNA as well as of TAT mRNA were localized predominantly in the periportal and intermediate zone after 60 h of starvation. PCK and TAT mRNA both decreased markedly during the first 2 h of refeeding and then remained almost constant. Whereas the alterations in the overall abundance of the two mRNAs were similar, the distribution patterns of both mRNAs differed. While PCK mRNA became more and more restricted to a small area of periportal cells towards the end of refeeding, TAT mRNA was first evenly distributed in the periportal and perivenous area with higher amounts in the intermediate zone and then again was predominantly located in the periportal area. The present data indicate that the predominant periportal localization of PCK and TAT activity and enzyme protein is regulated mainly at the pretranslational level.

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG--TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5' to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from a patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3' end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.

Just before birth, changes occur in the metabolic capacities of rat liver so that the animal can adapt to changes in the substrate supply. In utero, glucose is the main energy-generating fuel and the liver metabolism is directed towards glucose degradation. The activities of the rate-limiting enzymes of glycolysis, hexokinase and phosphofructokinase, are high. In preparation for post-natal life, when the continuous glucose supply from the mother is interrupted, very large amounts of glycogen are stored in the late fetal liver. With the intake of the fat-rich and carbohydrate-poor milk diet, the animal develops the ability to synthesize glucose de novo from non-carbohydrate precursors. During suckling, metabolic energy is derived mainly from the beta-oxidation of fatty acids, which in turn is an essential prerequisite for the high rate of gluconeogenesis, by yielding acetyl-CoA for the activation of pyruvate carboxylase and by generating a high NADH/NAD ratio for the shift of the glyceraldehyde 3-phosphate dehydrogenase reaction in the direction of glucose formation.--The developmental adaptation of metabolism and the process of enzymatic differentiation are closely connected with the maturation of the endocrine system and the changes in the concentration of circulating hormones. The neonatal regulation of phosphoenolpyruvate carboxykinase and of tyrosine aminotransferase by variations in the hormonal milieu around birth, and also the interaction of hormonal and nutritional factors in the induction of serine dehydratase and glucokinase at the end of the suckling period, will be discussed in detail.

Freshly isolated liver parenchymal cells were maintained in either short-term monolayer, suspension of long-term monolayer culture. Rapidly occurring processes through hepatocellular membrane, e.g., the enhanced amino acid transport and the concomitantly increased potassium influx following progressive starvation, were kinetically evaluated best in short-term monolayer culture. The inducibility of tyrosine aminotransferase by glucagon, dexamethasone, and a combination of both was compared in suspension and in monolayer culture. The induction of slowly inducible foreign compound-metabolizing enzymes, (e.g., ethoxycoumarin-O-dealkylase, p-nitroanisole-O-demethylase, and UDP-glucuronyltransferase) by phenobarbital, 3-methylcholanthrene, and dexamethasone were studied in long-term monolayer culture. The latter system was also used to maintain isolated kidney cortical tubules for the investigation of renal enzyme adaptation during progressive time in culture.

Some effects of culturing adult rat hepatocytes on each of four different substrates--laminin (LN), collagen type I (C-I), collagen type IV (C-IV), and fibronectin (FN)--have been investigated under defined conditions. No differential effect on the attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate on which they had been cultured. After 14 d of culture, gamma-glutamyltranspeptidase activity was highest in cells cultured on C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes cultured in serum-free medium.

Salivary glands incubated in various concentrations of pyridoxine (Vitamin B6) show increasing tyrosine aminotransferase (TAT) activity at concentrations up to 10(-5) M and then decreasing activity up to 10(-2) M but in all cases the activity is greater than that of the controls. This increase in activity is demonstrable for up to 6 h, the longest period tested, and is dependent on the synthesis of new mRNA. A similar increase in TAT activity is observed in salivary glands subjected to heat shock. Antibodies prepared against purified tyrosine aminotransferase precipitate a peptide of the same molecular weight (40 KD) as that induced by pyridoxine.

Most cirrhotics have tyrosinemia and subnormal tyrosine tolerance; in some the ability to metabolize p-hydroxyphenylpyruvic and homogentisic acids is impaired. In previous studies, the initial transamination appeared to be the rate-limiting step. In this study, hepatic tyrosine transaminase activity was compared in liver biopsies from eight noncirrhotic and ten cirrhotic subjects to determine whether the subnormal tyrosine tolerance was related to decreased maximal activity of this enzyme. Fasting plasma tyrosine in the cirrhotics (133 +/- 43 micromol/liter) was significantly higher (P less than 0.005) than in the noncirrhotic subjects (64 +/- 25 micromol/liter). Tyrosine transaminase activity in the cirrhotic livers (42 +/- 11 micromol PHPA/g liver/hr, or 0.47 +/- 0.1 micromol PHPA/mg protein/hr) was not significantly different from the enzyme activity in the noncirrhotic liver (43 +/- 7 micromol PHPA/g liver/hr, or 0.39 +/- .12 micromol PHPA/mg protein/hr.) Thus elevated tyrosine levels in cirrhotics cannot be explained by decreased tyrosine transaminase activity in the liver, and other explanations must be sought.

Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture. Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture. Especially C-5, 6, and 9 showed excellent efficiency for such action. The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation. The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media. Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes. The addition of dexamethasone (10 microM) caused a 1.7 to 2.1-fold induction in TAT activity. The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.

Tyrosine aminotransferase (TAT; EC 2.6.1.5) is a liver enzyme involved in amino acid metabolism and gluconeogenesis. Low levels of TAT have been implicated in several inherited disorders, particularly tyrosinemia II (Richner-Hanhart syndrome). We have determined the chromosomal location of the human TAT gene by Southern blot hybridization analysis of DNAs from 18 human X rodent hybrid cell lines, using a rat cDNA probe. The results indicate that the TAT gene maps to chromosome 16. Analysis of two hybrids containing a rearranged chromosome 16 allowed assignment of the TAT locus to 16q22----24. In situ hybridization to human metaphase chromosomes confirmed this regional assignment.

The enhancement of L-tyrosine aminotransferase activity by dexamethasone, an exclusive function of the liver, was serially measured at different passages of eight rat liver epithelial cell lines initiated and continuously grown in either a serum-supplemented medium or a serum-free medium. The enzyme basal activity was found to be 5.4 +/- 1.8 mU for cell lines in serum and 6.8 +/- 3.4 mU for cell lines without serum. Under the influence of dexamethasone (10(-6) mol/l for 5 hours) this basal level could be increased up to 2.9 fold in the presence of serum and 2.5 fold in its absence when investigations were carried out at early passages. During the following subcultures the induction ratio gradually declined and scarcely any induction could be detected after the 15th passage for cells grown in serum and after the 25th passage for cell lines grown without serum.

The effects of dexamethasone on multiple metabolic functions of adult rat hepatocytes in monolayer culture were studied. Adult rat liver parenchymal cells were isolated by collagenase perfusion and cultured as a primary monolayer in HI/WO/BA, a serum free, completely defined, synthetic culture medium. Cells inoculated into the culture medium formed a monolayer within 24 hr. Electron microscopy showed that the cells in primary culture had a fine structure identical to liver parenchymal cells in vivo, including the observation of desmosomes and bile canaliculi in intercellular space. There was significant gluconeogenesis by the cells 24 hr postinoculation but it had decreased markedly by 48 hr. There was a marked induction of tyrosine aminotransferase (TAT) by dexamethasone, which was maintained for up to 72 hr postinoculation of cells. The transport of alpha-aminoisobutyric acid into the cells in monolayer culture was stimulated by dexamethasone and was dependent on the concentration of dexamethasone. Albumin synthesis and secretion by the cells was measured by a quantitative electroimmunoassay. Albumin production was shown to increase linearly over an incubation period of 24 to 48 hr postinoculation. Dexamethasone depressed the albumin synthesis. The effects of dexamethasone are slow, and at times require more than 6 hr to show variation from the control, indicating that dexamethasone is not a single controlling hormone. Possibly it functions in a cooperative and coordinating role in the regulation of cell metabolism.

In experiments on glucose-6-phosphatase and tyrosine aminotransferase it was shown that radiation induces changes in enzymic differentiation in perinatal rat liver. A study was made of the probable reasons for the observed changes. It was shown that the macromolecular system of the protein enzyme synthesis was not damaged by the radiation doses used. The observed decrease in glucose-6-phosphatase activity during late embryogenesis, after pre-irradiation at early organogenesis, is eliminated by administration of exogenous thyroxine. A radiation-induced rise in the tyrosine aminotransferase activity during the perinatal period correlated with the cyclic AMP system status. It is proposed that modification of enzymic differentiation after irradiation results from the change in the amount of inductors.

Insulin is able to stimulate a growth response in a variety of different cell types. However, the role of the insulin receptor in mediating this response is not clear. Indeed, it has been reported that the ability of insulin to stimulate a growth response is a result of its interaction with other growth factor receptors rather than the insulin receptor. We have previously reported that the H-35 hepatoma cell line responded to physiological concentrations of insulin as a growth factor and that the relative potency of proinsulin suggested that this response was mediated by the insulin receptor. In this report, two experimental approaches are used to demonstrate the involvement of the insulin receptor in mediating the growth response. Two different preparations of antibody to the insulin receptor are found to be capable of stimulating this response. In addition, the human insulin-like growth factors (IGF-I and II) show very low cross-reactivity with the insulin receptor and are significantly less potent than insulin in stimulating the growth response.

Summary: We determined the activities of tyrosine aminotransferase (TAT, EC 2.6.1.5), p-hydroxyphenylpyruvate oxidase (p-HPPA oxidase, EC 1.14.2.2) and fumarylacetoacetate fumarylhydrolase (FAH, EC 3.7.12) in cytosol of the liver and kidney tissues obtained at autopsy from a case of hereditary tyrosinemia type I. Values were compared with those from a control group of autopsied tissues from three adults and six children, who had died of other causes. In tyrosinemia, these three hepatic enzyme activities were all decreased: TAT showed approximately 35%, p-HPPA oxidase 11%, and FAH 60% of the corresponding control values. On the other hand, kidney enzymes in tyrosinemia revealed that FAH was most significantly decreased to approximately 14% of the control activity. Km values for substrate— determined for p-HPPA oxidase and FAH—were not different between the patient and controls, suggesting no altered properties of these enzymes. We conclude that in the present case of hereditary tyrosinemia type I, the activities of p-HPPA oxidase in liver and FAH in kidney were most strikingly affected. This fact may in part explain the deteriorated metabolism of tyrosine observed in this patient.

The stability of the expression of six differentiated functions was examined during long-term cultivation of rat hepatoma cells. Faza 967 cell line--a clonal descendant of the Reuber H35 hepatoma--is characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenetic enzymes; secretion of serum albumin; and the presence of liver isozymes of alcohol dehydrogenase (ADH-L), aldolase (aldolase-B) and five isozymes of lactate dehydrogenase (LDH). During the 3-year-long cultivation of Faza 967 cells TAT specific activity, inducibility, and albumin production were reduced drastically whereas the expression of the three liver-specific isozymes examined was maintained. The majority of Faza 967 cells were able to perform gluconeogenesis after 3 years of continuous cultivation. Our results show that long-term cultivation of hepatoma cells may change the expression of certain liver-specific functions independently of the expression of other differentiated functions.

A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and pseudodiploid clone derived from adult rat liver cell line were positive for alpha-fetoprotein.

Two Fischer 344 rat hepatoma cell strains, JM1 and JM2, have been isolated from a primary hepatocellular carcinoma. Primary tumor formation was induced in a two-thirds partially hepatectomized rat by a single low dose (70 mg/kg of diethylnitrosamine followed by chronic phenobarbital administration (0.1 g/100 ml drinking water). The primary tumors were passed three times by subcutaneous implantation of tumor fragments into the inguinal region of syngeneic recipients. The fourth pass was by injection of tumor cells directly into the livers of recipient rats. Several weeks later, the tumor-containing rat livers were subjected to collagenase perfusion. Two cell lines emerged from tissue culture of the cells isolated by perfusion. Each cell line was cloned by serial dilution. Cells JM1 and JM2 were tumorigenic when injected into syngeneic rats. The tumors, which arose from injected cell strains, exhibited several characteristics of hepatocellular carcinoma. Morphology was examined by light and electron microscopy. Histochemical studies of JM1 and JM2 cells grown in vitro and in vivo were done. The levels of tyrosine aminotransferase and three microsomal enzymes of importance to drug and carcinogen metabolism were investigated. To our knowledge, this is the first report of cell strains derived from an initiation promotion protocol in rats.

An improved model describing receptor/gene-mediated pharmacodynamics of prednisolone is presented which consists of seven differential equations. Data for plasma prednisolone concentrations, free hepatic glucocorticoid receptors, and hepatic tyrosine aminotransferase activity (TAT) following low (5 mg/kg) and high (50 mg/kg) doses of prednisolone are used to quantitate the kinetics and dynamics of this synthetic steroid in the rat. In contrast to the earlier model, the newer model provides for a coupling and simultaneous fitting of receptor and TAT data and is able to describe the recycling of receptors between cytosol and nucleus and the return of cytosolic receptors to baseline following glucocorticoid elimination. A numerical technique to determine the efficiency of TAT induction based on area under the curve calculations is presented, which supports the hypothesis that nonlinear dose-response effects are due to dose and time-dependent receptor depletion in the cytosol. Simulations are presented to examine the major determinants of corticosteroid effects and to compare the effects of single- and multiple-dose regimens in maximizing drug effects.

L-Tryptophan increases the activity of hepatic amino acid metabolizing enzymes, affects gluconeogenesis and displays a modulatory effect on several enzymes connected with RNA synthesis. The underlying mechanism differ in individual cases and result in both an increase of enzyme synthesis de novo and a decrease of enzyme degradation. Tryptophan displays a unique effect causing aggregation of hepatic polyribosomes connected with enhanced protein synthesis and preceded by a higher transport of poly (A) messenger RNA from the nucleus to the cytoplasm. The variety of rather specific effects mediated by tryptophan brings to mind hormonal action and the existence of specific tryptophan receptors is predicted.

Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.

Isolated liver cells, which were prepared from adult rats by a trypsin-liver-perfusion technique, were treated with dexamethasone or hydrocortisone at a concentration of 7.7 X 10(-6) M for 8 days in primary culture. The treated cultures displayed homogeneous population consisting of epithelial-like clear cells, while the untreated cultures displayed mixed population consisting of epithelial-like clear cells and fibroblast-like cells. The epithelial-like clear cells, which proliferated in the cultures treated with glucocorticoids for 8 days in primary culture, did not show any morphological changes following cultivation in glucocorticoid-free medium. After continuous glucocorticoid-treatment for more than 1 month, the treated cultures showed relatively low cell densities at confluence. The surface area of individual epithelial-like clear cells in the cultures treated with glucocorticoids for long periods of time was evidently greater than that in the cultures treated for only 8 days. The epithelial-like clear cells had glucose 6-phosphatase and tyrosine aminotransferase activities even though the levels of these enzyme-activities were very low compared with those in rat liver homogenates.

Influence of Age on ortho -Hydroxyphenylacetic Acid Excretion in Phenylketonuria and Its Genetic Variants

A pharmacokinetic/pharmacodynamic model describing receptor-mediated effects of prednisolone is presented. The basis of the model is the generally accepted mechanism of action of steroid hormones in which corticosteroids bind to cytosolic receptors forming steroid-receptor complexes, which are activated and translocated into the nucleus. There the complexes associate with specific DNA sequences and modulate the rate of transcription of DNA into specific RNAs that code for the synthesis of proteins that elicit biological responses. Prednisolone, 5 or 50 mg/kg, was administered intravenously to adrenalectomized rats. Total plasma, free plasma, CBG-free plasma, and liver prednisolone concentrations were measured simultaneously with free hepatic cytosolic glucocorticoid receptor concentrations and tyrosine aminotransferase (TAT) activity of the liver as a function of time. The association/dissociation kinetics of prednisolone binding to the glucocorticoid receptor were measured separately in vitro at 37 degrees C. Total plasma, free plasma, and CBG-free plasma prednisolone concentrations could be used equally well in the model to account for the time course of receptor concentrations and TAT activity. However, use of liver steroid concentrations resulted in an overestimation of receptor depletion. Steroid concentrations in plasma increased 20 to 30-fold with a tenfold increase in dose, but receptor occupancy and TAT activity over time increased about threefold. While prednisolone pharmacokinetics were dose-dependent, parameters describing receptor kinetics and TAT activity were constant at each prednisolone dose. The major determinants of receptor-mediated glucocorticoid activity are confirmed to be the availability of the receptor, drug-receptor dissociation rate, and corticosteroid persistence in the biophase.

Chronic Tyrosinemia Associated with 4-Hydroxyphenylpyruvate Dioxygenase Deficiency with Acute Intermittent Ataxia and without Visceral and Bone Involvement

A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O2:5% CO2 atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals.

The liver enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5) catalyzes the rate-limiting step in the catabolic pathway of tyrosine. Deficiency in TAT enzyme activity underlies the autosomally inherited disorder tyrosinemia II (Richner-Hanhart syndrome). Using a human TAT cDNA clone as hybridization probe, we have determined the chromosomal location of the TAT structural gene by Southern blot analysis of DNAs from a series of human X rodent somatic cell hybrids. The results assign the TAT gene to human chromosome 16.

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by L-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by L-phenylalanine. Prephenate dehydratase was strongly activated by L-tyrosine. NAD+-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by L-tyrosine. Two aromatic aminotransferases were resolved, one preferring the L-phenylalanine:2-ketoglutarate substrate combination and the other preferring the L-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which L-tyrosine modulates L-phenylalanine synthesis via activation of prephenate dehydratase. L-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.