Ageing and the Regulation of Cell Activities during Exposure to Cold

The inability to maintain body temperature and a selective pattern of changes in the regulation of cell activities were revealed by briefly exposing ageing C57B1/6J male mice to cold (10°C). The induction of liver tyrosine aminotransferase (TAT) during exposure to cold (a gene-dependent process) was markedly delayed in senescent mice (26 months old) as compared with younger mice (3–16 months old); after the delay, the rate of increase of TAT was similar to that prevailing in younger mice. Direct challenge of the liver with injections of corticosterone or insulin elicited the induction of TAT on an identical time course in young and senescent mice. These experiments provide an example of an age change in a gene-dependent cell process (the delayed induction of TAT in senescent mice during exposure to cold) which is not due to a change in the potential of the genome for responding when exogenous stimulae are supplied (injection of hormones). In contrast to the age-related change in liver cell activities, no significant changes were found in the secretion of corticosterone during exposure to cold. Although the seat of these selective age-related changes in the regulation of cell activities remains unclear, it is argued that generalized damage to the genome of cells throughout the body is not involved. The results of this and other studies showing the selective effect of age on cell activities are considered in terms of the concept that many cellular age changes represent the response of cells to primary age-related changes in humoral factors in the internal environment of the body.     

Mechanism of a decrease of inducibility of tyrosine aminotransferase by carcinogenic aminoazo dyes. Absence of direct influence of o-aminoazotoluene on the enzyme molecules

The effect of o-aminoazotoluene (OAT) on the activity of tyrosine aminotransferase (TAT) from mouse liver cytosol under its incubation in the presence of the systems providing for the metabolic activation of the cancerogen (liver microsomes and NADPH2) and dephosphorylation of TAT molecules (light mitochondria and ATP) was studied. It was shown that OAT has neither direct nor indirect (via the phsophorylation--dephosphorylation systems) effect on the activity of TAT. It was concluded that the decrease of TAT induction by hydrocortisone in vivo resulting from injection of OAT to the mice is not due to the direct influence of the cancerogen on the enzyme molecules.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Tyrosine aminotransferase induced in cells genetically and epigenetically deficient for this enzyme

Mice homozygous for lethal deletions around the albino (c) locus are deficient for several liver-specific enzymes, including tyrosine aminotransferase (TAT). The structural gene coding for this enzyme appears to be intact in these mutants and can be “activated” in homozygous mutant mouse liver-rat hepatoma cell hybrids. The present study demonstrates that the mouse form of TAT can also be induced in both normal and mutant mouse skin-rat hepatoma cell hybrids. Thus, a liver-specific enzyme is expressed in skin cells, both normal and mutant, the normal differentiated state of which excludes TAT expression.     

Measurement of brown adipose tissue uncoupling protein by enzyme linked immunosorbent assay

Antibody to uncoupling protein (UCP) purified from rat brown adipose tissue (BAT) was raised in rabbits and an enzyme linked immunosorbent assay was developed. The antiserum did not cross-react with other mitochondrial proteins from BAT and from other tissues but cross-reacted with UCP from hamster, guinea pig and mouse. The assay is capable of detecting 5 ng of UCP. Using this assay and a crude mitochondrial preparation, UCP content of BAT was shown to increase during cold adaptation.     

The increase in hepatic tyrosine aminotransferase activity by ethanol administration involves an acceleration of enzyme synthesis

The present study was conducted to examine the nature of the increase in tyrosine aminotransferase (TAT) activity by acute ethanol administration. A significant rise in aminotransferase activity was observed as early as 1 hr after intact rats were gavaged with ethanol. Ethanol administration also increased TAT activity in adrenalectomized rats. Inhibition of ethanol metabolism by pyrazole administration had no effect on the ethanol-induced increase in TAT activity. Immunochemical analyses revealed that the enhancement of TAT activity in ethanol-fed rats correlated with an increase in aminotransferase protein. Measurement of the rate of TAT synthesis showed that in ethanol-fed rats, [3H]leucine was incorporated into the aminotransferase protein at a higher rate than in controls by a factor which was similar to the enhancement in enzyme activity. Our findings indicate that an acceleration of TAT synthesis fully accounts for the increase in TAT activity during the early stage of enzyme induction. TAT induction by ethanol administration is not dependent upon an increase in adrenal corticosteroid production, nor does it require ethanol metabolism.     

Daily Rhythms of Core Temperature and Locomotor Activity Indicate Different Adaptive Strategies to Cold Exposure in Adult and Aged Mouse Lemurs Acclimated to a Summer-Like Photoperiod

Daily variations in core temperature (Tc) within the normothermic range imply thermoregulatory processes that are essential for optimal function and survival. Higher susceptibility towards cold exposure in older animals suggests that these processes are disturbed with age. In the mouse lemur, a long-day breeder, we tested whether aging affected circadian rhythmicity of Tc, locomotor activity (LA), and energy balance under long-day conditions when exposed to cold. Adult (N?=?7) and aged (N?=?5) mouse lemurs acclimated to LD14/10 were exposed to 10–day periods at 25 and 12°C. Tc and LA rhythms were recorded by telemetry, and caloric intake (CI), body mass changes, and plasma IGF-1 were measured. During exposure to 25°C, both adult and aged mouse lemurs exhibited strong daily variations in Tc. Aged animals exhibited lower levels of nocturnal LA and nocturnal and diurnal Tc levels in comparison to adults. Body mass and IGF-1 levels remained unchanged with aging. Under cold exposure, torpor bout occurrence was never observed whatever the age category. Adult and aged mouse lemurs maintained their Tc in the normothermic range and a positive energy balance. All animals exhibited increase in CI and decrease in IGF-1 in response to cold. The decrease in IGF-1 was delayed in aged mouse lemurs compared to adults. Moreover, both adult and aged animals responded to cold exposure by increasing their diurnal LA compared to those under Ta?=?25°C. However, aged animals exhibited a strong decrease in nocturnal LA and Tc, whereas cold effects were only slight in adults. The temporal organization and amplitude of the daily phase of low Tc were particularly well preserved under cold exposure in both age groups. Sexually active mouse lemurs exposed to cold thus seemed to prevent torpor exhibition and temporal disorganization of daily rhythms of Tc, even during aging. However, although energy balance was not impaired with age in mouse lemurs after cold exposure, aging was associated with lower LA and Tc during the night and delayed decrease in IGF-1. This might reflect that adaptive strategies to cold exposure differ with age in mouse lemurs acclimated to a summer-like photoperiod.     

Tyrosine aminotransferase and gamma-glutamyl transferase activity in human fetal hepatocyte primary cultures under proliferative conditions

The ontogeny of gamma-glutamyl transferase (GGTase; E.C.2.3.2.2) and tyrosine aminotransferase (TAT; E.C.2.6.1.5) activities in 14 to 36 weeks gestational and neonatal hepatocytes during development of human fetal liver was studied. Subsequently, 20-24 weeks gestational hepatocytes were cultured in media supplemented with epidermal growth factor (EGF) and insulin with or without glucagon and dexamethasone to investigate the proliferation and differentiation of fetal hepatocyte in vitro using GGTase and TAT as biochemical markers. During the development of the liver, the activity of GGTase increased continuously from the first trimester through the third trimester and decreased (p < 0.001) in neonates. A low basal level of TAT activity was seen only during the third trimester, which then increased significantly (p < 0.001) in neonates. Fetal hepatocytes, in the presence of EGF and insulin, undergo proliferation from the fourth to 10th day with an increase in cell number (p < 0.001) and concomitant increase (p < 0.001) in GGTase activity. As the cells attain confluence, enzyme activity decreased significantly (p < 0.001) from the 10th to 16th day. Maximal TAT activity (p < 0.001) was observed at 48 h of culture, which decreased, but not significantly, during cell proliferation and the enzyme activity was regained as the cultures attained confluence. Furthermore, TAT activity was induced synergistically (p<0.001) in the presence of glucagon and dexamethasone, while GGTase was inhibited (p<0.001). These results indicate that GGTase increases with proliferation, whereas TAT, once it has been expressed, is not suppressed during cell proliferation. In conclusion, human fetal hepatocytes undergo enzymic differentiation by 48 h of culture, and proliferate with an increase in GGTase in the presence of growth factors with maintenance of differentiated status up to the studied 16 days of culture.     

Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures

Time-course changes in rosmarinic acid (RA) formation and activities of tyrosine aminotransferase (TAT) isoforms were examined in Anchusa officinalis suspension cultures. Three TAT isoforms (TAT-1, TAT-3, TAT-4) were resolved by Mono-Q anion-exchange column chromatography. The proportion of the TAT-3 activity within the total TAT activity remained high regardless of the growth stage of the cultured cells. TAT-1 activity was positively correlated with the rate of RA biosynthesis during linear growth stage of the culture cycle, while TAT-4 activity was rapidly induced in conjunction with transfer to fresh medium coincident with a transient increase in RA synthesis. Based on these results, as well as the substrate specificity of each TAT isoform, it was concluded that both TAT-1 and TAT-4 are closely involved in RA biosynthesis. TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate, and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.Abbreviations PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid     

Isolation and properties of cortisol inducible and cortisol non-inducible isoenzymes of rat liver tyrosine aminotransferase]

Rat liver contains two groups of tyrosine aminotransferase (TAT) isoenzymes; during electrophoresis in agar gel one of the groups moves to the anode and the other--to the catode. Cortisol is shown to induce only the anode isoenzymes of TAT, which were isolated, purified and thoroughly analyzed. The inducible anode isoenzyme of TAT spearated from other proteins is more sensitive to the effect of proteases (trypsin and chymotrypsin) than the catode isoenzyme. Some kinetic parameters of the purified TAT isoenzymes were studied. Both isoenzymes have pH optimum around 7.5; their apparent Km values for tyrosine are also similar. However, the catode isoenzyme of TAT possesses a higher affinity for alpha-ketoglutarate than does the anode isoenzyme. Unlike the latter, the former isoenzyme may use oxaloacetate as an amino group acceptor. Pyridoxal phosphate is firmly bound to the catode isoenzyme and can be readily spearated from the anode isoenzyme during dyalisis. An increased sensitivity of the inducible isoenzyme to proteases is due not only to the possibility of coenzyme dissociation, but also to some specific properties of the apoenzyme. The results obtained support the assumption that a high sensitivity of the inducible isoenzymes to proteases provides for a removal of excessive amounts of the enzymes from the cells under cessation of hormonal induction, thus maintaining enzymatic homostasis in the cell.     

Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway

Basolateral efflux is a necessary step in transepithelial (re)absorption of amino acids from small intestine and kidney proximal tubule. The best characterized basolateral amino acid transporters are y+LAT1-4F2hc and LAT2-4F2hc that function as obligatory exchangers and thus, do not contribute to net amino acid (re)absorption. The aromatic amino acid transporter TAT1 was shown previously to localize basolaterally in rat's small intestine and to mediate the efflux of L-Trp in the absence of exchange substrate, upon expression in Xenopus oocytes. We compared here the amino acid influx and efflux via mouse TAT1 in Xenopus oocytes. The results show that mTAT1 functions as facilitated diffusion pathway for aromatic amino acids and that its properties are symmetrical in terms of selectivity and apparent affinity. We show by real-time RT-PCR that its mRNA is highly expressed in mouse small intestine mucosa, kidney, liver, and skeletal muscle as well as present in all other tested tissues. We show that mTAT1 is not N-glycosylated and that it localizes to the mouse kidney proximal tubule. This expression is characterized by an axial gradient similar to that of the luminal neutral amino acid transporter B0AT1 and shows the same basolateral localization as 4F2hc. mTAT1 also localizes to the basolateral membrane of small intestine enterocytes and to the sinusoidal side of perivenous hepatocytes. In summary, we show that TAT1 is a basolateral epithelial transporter and that it can function as a net efflux pathway for aromatic amino acids. We propose that it, thereby, may supply parallel exchangers with recycling uptake substrates that could drive the efflux of other amino acids.     

Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity

The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48-60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48-60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30-45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48-60) had no effect. Importantly, administration of the penetratin or TAT(48-60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin-siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.     

Unlinked regulation of the sensitivity of primary glucocorticoid-inducible responses in mouse mammary tumor virus infected Fu5-5 rat hepatoma cells

The enzyme tyrosine aminotransferase (TAT) is induced by unusually low concentrations of glucocorticoids in Fu5-5 cells. We have isolated clones of Fu5-5 cells infected with mouse mammary tumor virus (MMTV) in order to simultaneously compare the glucocorticoid regulation of the host cell gene, TAT, with that of another primary inducible gene, MMTV. In the two clones that were examined in detail, MMTV RNA induction occurred at 4- to 11-fold higher concentrations of dexamethasone than those needed for induction of TAT mRNA. Furthermore, the amount of agonist activity displayed by the irreversible antiglucocorticoid dexamethasone 21-mesylate was greater for the induction of TAT mRNA than for MMTV RNA. These results extend our previous observations of unequal sensitivity of induction of TAT enzyme activity in two hepatoma cell lines and show that differential glucocorticoid regulation of gene induction within the same cell can occur at a pretranslational step. The present data also indicate that the unusual properties of TAT gene induction are not shared by all primary, glucocorticoid-inducible responses of the same cell and imply that additional factors mediating differential regulation of glucocorticoid-responsive genes are involved.     

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast

Low‐temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low‐temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:776–783, 2014     

Factors Affecting Developmental Increase of Tyrosine Aminotransferase in Rat Liver Immediately after Birth

Labeling with ³⁵S-methionine of dispersed hepatocytes prepared from neonatal rat liver and successive immunoprecipitation with antiserum against tyrosine aminotransferase (TAT) indicated that increase of TAT activity to a peak about 12 hours after birth and the decrease thereafter are mainly due to changes of TAT synthesis. Similar changes of TAT activity was also observed in the livers of premature neonates which were taken out by Caesarian section and nursed by foster mothers. This indicated that the appearance of TAT activity at this period is not an event programmed along with fetal development but is triggered by birth. The level of glucagon in neonatal plasma increased after birth. Administration of glucagon to neonates caused a great increase of TAT activity whereas the effect of dexamethasone was not so evident. These suggested that glucagon is an important factor affecting the abrupt appearance of TAT after birth.