Involvement of sequences near both amino and carboxyl termini in the rapid intracellular degradation of tyrosine aminotransferase.
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The degradation of rat liver tyrosine aminotransferase has been studied after transfection of suitable expression vectors into mammalian cells in culture. A normal rapid rate of degradation (half-life about 6 h) was observed in cells under stable transfection conditions. However, the higher enzyme levels produced during transient transfections or after amplification with methotrexate caused the apparent half-life of degradation to increase substantially. Analysis of expression in Chinese hamster ovary (CHO)-DG44 cells from vectors with deletions near either end of the tyrosine aminotransferase coding sequence showed that approximately the first 40 and the last 12 amino acid residues are not required to obtain normal catalytic function. When catalytically active deletion mutants were examined for effects on tyrosine aminotransferase degradation in stably transfected CHO-DG44 cell populations, short sequences near each end of the protein were found to be necessary for rapid degradation. The required sequence near the amino terminus is located between amino acids 30 and 40 and includes the highly basic region RKKGRKAR, a potential ubiquitin attachment site. The other essential sequence (EECDK) is located at the very COOH terminus of the 454-amino acid chain and is part of an acidic domain rich in cysteines and having PEST characteristics (rich in Pro, Glu, and Thr). Ser448, a potential casein kinase II phosphorylation site, is not required for activity or rapid degradation of tyrosine aminotransferase. No correlation was observed between the intracellular degradation rates of the various mutant proteins and their heat stabilities in vitro. (+info)
Selective loss of a DNase I hypersensitive site upstream of the tyrosine aminotransferase gene in mice homozygous for lethal albino deletions.
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Several overlapping chromosomal deletions spanning the albino locus in the mouse cause perinatal lethality when homozygous and a block in the transcriptional induction of various unlinked hepatocyte-specific genes. Studies of such lethal albino deletion homozygotes in perinatal stages revealed a deficiency in the transcriptional inducibility of the tyrosine aminotransferase (TAT) gene by glucocorticoids; yet, glucocorticoid receptor and hormone levels were shown to be unaffected. To identify a molecular defect underlying the failure of inducible expression, we examined the chromatin structure of the TAT gene. Whereas in wild-type animals the TAT promoter becomes DNase I hypersensitive at birth, such hypersensitivity fails to develop in lethal albino deletion homozygotes. By contrast, the deletions do not affect the appearance of three DNase I-hypersensitive sites upstream of the TAT promoter in the liver, nor do they affect two hypersensitive sites upstream of the expressed alpha-fetoprotein gene. These findings demonstrate that the abnormality of chromatin structure identified in lethal albino deletion homozygotes occurs on a highly selective basis. Specifically, normal differentiation of the TAT promoter chromatin appears to depend directly or indirectly on the action and product of a gene mapping within the deleted region. (+info)
Mechanisms for glucocorticoid inhibition of immediate hypersensitivity reactions in rats.
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The inhibitory mechanisms of immediate hypersensitivity reactions by glucocorticoid (GC) were studied in rats. Homologous passive cutaneous anaphylaxis (PCA) mediated by IgE antibodies and cutaneous reactions caused by histamine, serotonin and leukotriene C4 were elicited at the same time in the same rats. Three kinds of GC, hydrocortisone, prednisolone and dexamethasone, inhibited all these reactions significantly. Although mediator-induced cutaneous reactions were inhibited transiently around 2 hours after GC administration, inhibition of PCA was more potent and lasted longer. A time lag seemed to be essential for both inhibitions. IgE antibody-mediated histamine release in vivo in the rat peritoneal cavity was also inhibited by GC administration significantly, and the inhibition was long lasting when compared to those of the mediator-induced cutaneous reactions. Tyrosine amino-transferase (TAT) activity in the rat liver increased significantly by GC administration, and the increased TAT activity was completely abrogated by simultaneous administration of 5 mg/kg of cycloheximide (CH). In the same experimental condition, although inhibition of histamine-induced cutaneous reaction by GC was completely abrogated, the inhibition of PCA elicited at the same time in the same rats was only partially attenuated. Furthermore, the same dose of CH little affected the dexamethasone inhibition of histamine release in the rat peritoneal cavity, although the increase of TAT activity in the liver of the same rats was completely abrogated. These results demonstrate that PCA is inhibited by GC through at least 2 mechanisms, inhibition of mediator release from mast cells and non-specific inhibition of vascular permeability increase caused by released mediators. Although the latter action of GC is dependent upon protein synthesis, the former seems to be mediated by a unique mechanism independent of protein synthesis. (+info)
Interactions of corticosterone, 5alpha-dihydrocorticosterone and dexamethasone with proteins in rat-liver cytosol.
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The intracellular binding of [3H]corticosterone and [3H]dexamethasone and their metabolites to macromolecules in rat liver cytosol was studied in vivo and in vitro. The macromolecules binding corticosterone and its metabolites were characterized as (a) a steroid conjugate-binding (Stokes radius 2.5 nm and sedimentation coefficient 4.1 S in high ionic strength; pI 8.7, (b) transcortin and (c) a glucocorticoid "receptor". Competition experiments indicate that corticosterone and dexamethasone bind to the same site of the glucocorticoid receptor molecule. Different Stokes radii between the corticosterone-receptor and the dexamethasone-receptor complexes (6.9 and 6.3 nm, respectively, in high ionic strength) indicate that the two ligands induce different conformations of the receptor protein. This may be of importance when explaining the qualitative differences between the cellular effects of natural and synthetic glucocorticoids. 5alpha-Dihydrocorticosterone, on the other hand, competed to a very limited extent with dexamethasone for binding sites on the receptor. An assay of the inductive effect on liver tyrosine aminotransferase and tryptophan oxygenase indicated that 5alpha-dihydrocorticosterone was practically devoid of glucocorticoid activity. It is concluded that 5alpha-dihydrocorticosterone probably does not act as the mediator of corticosterone action in rat liver. (+info)
Repression of the tyrosine, lysine, and methionine biosynthetic pathways in a hisT mutant of Salmonella typhimurium.
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A comparison was made of the repressibility of certain enzymes in the tyrosine, methionine, and lysine biosynthetic pathways in wild-type Salmonella typhimurium and a hisT mutant. The results show that (i) tyrosine represses the synthesis of the tyrosine-sensitive 3-deoxy-D-arabino-heptulsonic acid 7-phosphate synthetase and the tyrosine aminotransferase to the same extent in a hisT mutant as in wild type and (ii) there is no detectable alteration in the extent to which methionine represses O-succinylhomoserine synthetase or in the extent to which lysine represses the lysine-sensitive beta-aspartokinase as a result of the hisT mutation. (+info)
Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences.
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The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood. (+info)
Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects.
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Glucocorticoids (GCs) are the most commonly used antiinflammatory and immunosuppressive drugs. Their outstanding therapeutic effects, however, are often accompanied by severe and sometimes irreversible side effects. For this reason, one goal of research in the GC field is the development of new drugs, which show a reduced side-effect profile while maintaining the antiinflammatory and immunosuppressive properties of classical GCs. GCs affect gene expression by both transactivation and transrepression mechanisms. The antiinflammatory effects are mediated to a major extent via transrepression, while many side effects are due to transactivation. Our aim has been to identify ligands of the GC receptor (GR), which preferentially induce transrepression with little or no transactivating activity. Here we describe a nonsteroidal selective GR-agonist, ZK 216348, which shows a significant dissociation between transrepression and transactivation both in vitro and in vivo. In a murine model of skin inflammation, ZK 216348 showed antiinflammatory activity comparable to prednisolone for both systemic and topical application. A markedly superior side-effect profile was found with regard to increases in blood glucose, spleen involution, and, to a lesser extent, skin atrophy; however, adrenocorticotropic hormone suppression was similar for both compounds. Based on these findings, ZK 216348 should have a lower risk, e.g., for induction of diabetes mellitus. The selective GR agonists therefore represent a promising previously undescribed class of drug candidates with an improved therapeutic index compared to classical GCs. Moreover, they are useful tool compounds for further investigating the mechanisms of GR-mediated effects. (+info)
Expressions of hepatic genes, especially IGF-binding protein-1, correlating with serum corticosterone in microarray analysis.
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Microarray technology was evaluated for usefulness in assessing relationships between serum corticosterone and hepatic gene expression. Nine pairs of female Swiss mice were chosen to provide a wide range of serum corticosterone ratios; cDNA microarray analysis (approximately 8000 genes) was performed on their livers. A statistical method based on calculation of 99% confidence intervals discovered 32 genes which varied significantly among the livers. Five of these ratios correlated significantly with serum corticosterone ratio, including tyrosine aminotransferase, stress-induced protein, pleiotropic regulator 1 and insulin-like growth factor-binding protein-1; the latter has a potential role in cancer development. Secondly, linear regression of gene expression vs corticosterone ratios was screened for those with r> or =0.8 (P<0.01), yielding 141 genes, including some known to be corticosterone regulated and others of interest as possible glucocorticoid targets. Half of these significant correlations involved data sets where no microarray ratio exceeded +/- 1.5. These results showed that microarray may be used to survey tissues for changes in gene expression related to serum hormones, and that even small changes in expression can be of statistical significance in a study with adequate numbers of replicate samples. (+info)