Gene expression for a novel protein RGPR-p117 in various species: the stimulation by intracellular signaling factors

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors.     

Effect of cobalt on heme biosynthesis in rat liver and spleen.

1. Succinate-cytochrome c reductase activity in rat liver decreased to about 60% of the control value after a single injection of cobalt or in a steady state of intoxication, but the activity in the spleen was unaltered. 2. Incorporation of radioactive glycine and 5-aminolevulinate into heme of the liver was markedly inhibited by cobalt treatment. 3. 5-Aminolevulinate synthase [EC 2.3.1.37] activity in the liver decreased to 40% of the control value 4 hr after cobalt injection, and completely recovered 20 hr later. Phenylhydrazine-induced 5-aminolevulinate synthase activity in the spleen was not decreased by cobalt injection. 4. Porphobilinogen synthase [EC 4.2.1.24] activity in the liver decreased and reached its minimum value (42% of the control) 12 hr after cobalt injection. On the other hand, the activity in the spleen showed a marked increase 24 hr after coblat injection. 5. Ferrochelatase [EC 4.99.1.1] activity in the liver was essentially unaltered by cobalt treatment, while the activity in the spleen was elevated dramatically after 24 hr. 6. Concentrations of cobalt after a single injection were about 0.3 mM and 0.03 mM in the liver and spleen, respectively. 7. Inhibitions of 5-aminolevulinate synthase and porphobilinogen synthase activities by cobalt in vitro were not as marked as expected from in vivo experiments.     

The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.     

Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity

The tissue distribution and the structural heterogeneity of the rat angiotensinogen mRNA have been investigated with the aid of a previously cloned cDNA as well as a genomic DNA for rat angiotensinogen as analytical probes. The angiotensinogen mRNA is expressed not only in the liver but also in various tissues including the brain, kidney, adrenal gland, ovary, and lung. The relative levels of the mRNA in the above tissues have been estimated to be 3-4, 20-30 (for the next three tissues), and around 100 times less than that in the liver, respectively. The mRNAs in both hepatic and extrahepatic tissues are encoded by a single gene in the rat genome. At least four different size classes of the angiotensinogen mRNA that start with a single 5' terminus and differ only in the lengths of their 3'-untranslated regions have been identified, and these multiple mRNA species are most likely generated by using the polyadenylation signals AAUAAA and AUUAAA found 10-30 nucleotides upstream from the four polyadenylation sites. Because the structures of these multiple mRNA species do not vary among the tissues of the liver, brain, and kidney, angiotensinogen synthesized locally is structurally identical to that produced in the liver and may have some biological roles independent of the circulating angiotensinogen, mainly derived from the liver. In addition, the sequence of the 5'-flanking region of the angiotensinogen gene has been determined, and some features common to other steroid hormone-responsive genes have been discussed.     

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.     

ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species

Fatty acid synthase (FAS), a key lipogenic enzyme, is expressed in the two major sites of fatty acid production in the body, that is, the liver and the adipose tissue. Surprisingly, the relative contribution of these sites to lipogenesis is highly variable among species. For example, besides the situation in rodents, where liver and fat are equally active, lipogenesis in some mammals such as the pig occurs principally in adipose tissue, whereas in avian species, the liver is the main lipogenic site. We addressed the question concerning the factors determining the site of fatty acid synthesis. We show that the expression of adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1) mRNA, but not SREBP-2, is linked to FAS protein content or activity in adipose tissues and livers of pig, chicken, and rabbit. Tissue differences in ADD-1/SREBP-1 mRNA expression between species were paralleled by commensurate variations in the nuclear concentration of SREBP-1 protein. Moreover, overexpression of ADD-1/SREBP-1 by adenoviral gene transfer induces FAS in chicken adipocytes, where lipogenesis is normally low. Conversely, the expression of a dominant negative form of ADD-1/SREBP-1 in pig adipocytes downregulates FAS expression.These results reinforce the role of ADD-1/SREBP-1 as a key regulator of lipogenesis, by extending its importance to nonrodent mammals and birds. Furthermore, they establish that differential expression of ADD-1/SREBP-1 is a key determinant of the site of fatty acid synthesis in the body.-Gondret, F., P. Ferré, and I. Dugail. ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. J. Lipid Res. 2001. 42: 106;-113.     

Activity of key enzymes of heme metabolism and cytochrome P-450 content in the rat liver in experimental rhabdomyolysis and hemolytic anemia

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.     

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Specific species and tissue differences for the gene expression of calcium-binding protein regucalcin

The existence and expression of gene encoding the Ca²⁺-binding protein regucalcin in various species and tissues were investigated with Southern and Northern hybridization analyses using regucalcin cDNA (0.9 kb of open reading frame). Genomic Southern hybridization analysis demonstrated that regucalcin gene was widely conserved among higher animals including human, monkey, rat, mouse, dog, bovine, rabbit and chicken. The gene was not found in yeast. The Northern blot analysis of poly (A)⁺RNAs extracted from the liver of various species showed that regucalcin mRNA was predominantly expressed in rat and mouse, although the expression was also seen in human, bovine and chicken. Furthermore, the enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG indicated that hepatic regucalcin concentration was most pronounced in rat as compared with that of guinea pig, mouse and chicken. These observations show that the gene expression of regucalcin and its protein synthesis is unique in the liver of rats, suggesting the existence of a specific mechanism in demonstrating regucalcin synthesis from gene.     

Effects of antihypertensive drugs on hepatic heme biosynthesis,and evaluation of ferrochelatase inhibitors to simplify testing of drugs for heme pathway induction

Effects of a series of antihypertensive drugs on the activity of δ-aminolevulinate synthase and on the formation of porphyrins and cytochrome P-450 were examined in the 18-day-old chick embryo liver in ovo. Hydralazine, pargyline, phenoxybenzamine, clonidine, and spironolactone were found to induce δ-aminolevulinate synthase in this system. These drugs therfore have the potential to precipitate clinical expression in human hereditary hepatic porphyrias and should be avoided or used with caution in patients with these disorders. Differential effects of these and other drugs were observed in the avian liver, in that δ-aminolevulinate synthase was more commonly induced thatn were porphyrins and cytochrome -450; the synthase was usually highest 6–12 h after injection, whereas porphyrins and cytochrome P-450 were highest at 24 h. Furthermore marked porphyrin accumulation was not seen with many drugs that induce σ-aminolevulinate synthase and cytochrome P-450 but was more characteristic of compounds that reduced the metabolism of protoporphyrin to heme, such as 1,4-dihydro-3,5-dicarbethoxycollidne (DDC) and high dose of hydralazine. A sensitive and convenient method to test for capacity to induce heme biosynthesis was adapted for use in the chick embryo liver. This employed a relatively small “priming” dose (0.25 mg) of DDC given with a drug being tested and a fluorometric assay of porphyrins in a liver homogenate obtained at 24 h. This simple method should facilitate screening for those drugs which induce the synthesis of δ-aminolevulinate synthase and/or cytochrome P-450 and are potentially dangerous to patients with hereditary hepatic porphyria.