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.     

Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

Summary Non-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

Radioimmunoassay of urinary 3 beta-hydroxy-5-cholenoyl glycine in hepatobiliary disease

A radioimmunoassay for 3 beta-hydroxy-5-cholenoyl glycine in human urine has been developed. The antiserum was elicited with the antigen in which the steroid hapten is linked to a bovine serum albumin through the C-19 position. The [125I]-tyrosine derivative of the hapten was used as radioligand. The standard curves were linear ranging from 10 to 320 ng/mL. The cross-reactivities with other bile acids were not detectable and below 0.3% with cholesterol. Sample preparation includes extraction of 3 beta-hydroxy-5-cholenoyl glycine from urine and solvolysis of the sulfates--main form present in urine. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine was 0.373 +/- 0.133 mumol/day in healthy adults. Urinary excretion of 3 beta-hydroxy-5-cholenoyl glycine increased in chronic liver dysfunction, hepatoma and obstructive jaundice in this order.     

Detection of immunoreactive human placental oxytocin and its contractile effect on the uterine muscle

Freshly obtained human placentas from various periods of gestation were quantitatively analysed for their immunoreactive oxytocin (OT) content and its biological activity was examined in a Magnus apparatus by utilizing rat uterus. The mean values for placental immunoreactive OT per gram tissue increased from the first to the second trimester, maintaining its high level to term. The total content of placental OT also increased continually from the beginning of pregnancy to term. Blood levels of estrogen stimulated neurophysin (ESN) and OT were concomitantly enhanced through gestation. Placental extract and synthetic OT showed similar peaks in the elution pattern of ion-exchange chromatography through a carboxymethyl cellulose column. Synthetic OT and placental extract induced marked uterine contraction in diestrous rats. However placental extract previously incubated with OT antiserum failed to induce this effect. Though detection of immunoreactive OT by immunoassay alone does not provide definite identification of pituitary and placental OT, the present study suggests that placental immunoreactive OT could have a contracting effect on the uterine muscle.     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.