Species-specific induction of angiotensinogen mRNA in transgenic mouse liver during acute phase reaction.
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Acute-Phase Reaction↗
Biomedical subjects
A Fukamizu
Publications and source records attributed to A Fukamizu.
Structure and expression of the mouse angiotensinogen gene.
Angiotensinogen is a precursor of the multifunctional octapeptide hormone, angiotensin II. We have isolated the overlapping clones containing angiotensinogen gene locus from C57BL/6 mouse genomic DNA library and analyzed them by restriction enzyme mapping. The gene exhibited a structural organization similar to those of the human, rat and balb/c mouse angiotensinogen genes. Using a genomic DNA fragment of the mouse angiotensinogen gene as a probe, we have investigated the tissue distribution of angiotensinogen messenger RNA (mRNA) in C57BL/6 mouse. The angiotensinogen mRNA was highest in the liver and detectable in such tissues as brain, kidney, submandibular gland, ovary and heart. However, it was undetectable in lung and spleen under the condition used. Optimal alignments of the 5'-flanking regions among the human, rat and mouse angiotensinogen genes disclosed several deletions in the mouse sequence. To assay the promoter activity, the 5'-flanking region of the mouse angiotensinogen gene was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, then transfected into different cultured cells. The angiotensinogen gene sequences elicited preferential expression of CAT activity when introduced into HepG2 cells derived from liver and 293 cells from kidney but not in HeLa cells from uterus, suggesting the presence of a cell type-specific promoter within the sequences. These findings on the structure and expression of the mouse angiotensinogen gene should prove useful in studying the function and control of the angiotensin.
Angiotensinogen↗
Comparative study of renin expression in the coagulating glands of C57BL/6 and Balb/c mice.
The comparative localisation of renin in the genital organs, especially in the coagulating glands of male mice of the strains C57BL/6 and Balb/c, was investigated using immunocytochemical, immunoelectron microscopical and Northern blot techniques. Dot-like reactions for renin, of varying diameters, were detected immunocytochemically in the epithelial cells of coagulating glands of C57BL/6 mice, but not in those of Balb/c mice. In both strains, many electron dense granules differing in content and morphology were observed in the epithelial cells of the coagulating glands. Crystalline materials were sometimes contained in these granules. Colloidal gold particles indicating the presence of renin were detected in the electron dense granules of C57BL/6 mice, in which they showed a heterogeneous distribution and were especially located on the crystalline structure. No positive reaction was detected in these crystalline structures in Balb/c mice. Renin mRNA was detected in the coagulating glands of both C57BL/6 and Balb/c mice by Northern blot analysis. However, the expression in C57BL/6 coagulating glands was stronger than that in Balb/c. These findings suggest that renin is synthesised and released in the coagulating glands.
Animals↗
Identification of a previously unrecognized production site of human renin.
We found expression of the renin gene in the intestine of human, mouse and the transgenic mouse in which the 3' flanking sequences of the human renin gene function as a tissue-specific promoter. A cotransfection analysis showed that the promoter is activated by the product of adenovirus E1A 13S mRNA in cells originated from extrarenal tissues.
Adenoviridae↗
Expression of the human angiotensinogen gene in transgenic mice and transfected cells.
We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.
Angiotensinogen↗
Human renin in transgenic mouse kidney is localized to juxtaglomerular cells.
To examine whether expression of human renin in the transgenic mouse kidney is regulated in a cell-specific manner, we have characterized monoclonal antibodies against human renin and determined the renin-production site by immunohistochemistry. By using a monoclonal antibody specific for human renin, A6-11-6, we demonstrated that human renin in the transgenic mouse kidney is localized to the juxtaglomerular cells of afferent arterioles.
Animals↗
Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum.
A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method. Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450. The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s. Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s. These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms. The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus. P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.
Amino Acid Sequence↗
A case of renin producing leiomyosarcoma originating in the lung.
A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.
Abdomen↗
Structure and sequence analysis of the human activin beta A subunit gene.
The cloned genomic DNA containing the human activin beta A subunit gene were analyzed by restriction endonuclease mapping, Southern blotting and DNA sequencing. The activin gene is composed of two exons interrupted by the 9-kb intron. The TATA, CCAAT and CT-stretch sequences were found in the 5'-flanking region of the gene. An intronic sequence contained SV40 enhancer core element in the vicinity of the exon 1. In the 3'-flanking region, we identified eight consensus polyadenylation sequences, five ATTTA motifs, CA element consisting of (CA)14, AP-1 binding site and two SV40 enhancer core elements. A dot matrix analysis revealed the high degree of conservation between the human and rat sequences within the 3'-flanking region, suggesting a possible functional significance.
Activins↗
Regulation of human renin and angiotensinogen genes.
We have constructed pUCSV0cat with low background of chloramphenicol acetyltransferase (CAT) activity and pUCSV3cat (positive control), both containing a SV40 polyadenylation signal 5' to the CAT-coding gene and to the SV40 promoter, respectively. Using this modified pUCSV0cat, we found that human embryonic 293 cells have the ability to activate the promoter of the human renin gene. In addition, we identified the cis-acting sequences responsible for cell-specific expression of the human angiotensinogen gene in its 5'-flanking region.
Angiotensinogen↗
Structure and expression of the human angiotensinogen gene. Identification of a unique and highly active promoter.
We have isolated the human angiotensinogen gene from a genomic library and determined the exon-intron junction sequences. The gene is 12 kilobases long and consists of five exons interrupted by four introns, as a single copy in the human genome. Of particular interest are the positions of the introns in the human angiotensinogen gene which are identical to those in the highly homologous human alpha 1-antitrypsin and alpha 1-antichymotrypsin genes, as well as rat and mouse angiotensinogen genes. Northern blot analysis showed that human hepatoma cells (HepG2) produce a large amount of angiotensinogen mRNA but not human glioma cells (T98G). To assay the promoter activity, the 1.3-kilobase genomic fragment containing the 5'-flanking region, first exon, and a part of first intron at positions -1222 to +44 was fused upstream to the chloramphenicol acetyltransferase gene, then transfected into HepG2 and T98G cells. The gene sequence was active only in HepG2 cells, suggesting the presence of a functional promoter. Analysis of deletion mutants demonstrated that the 76-base pairs region from -32 to +44 containing the TATA box and first exon is the minimal promoter, whose activity is as high as that of the SV40 enhancer-promoter. Since the basal expression of the human angiotensinogen gene is much higher in HepG2 than T98G cells, these results may reflect cell-specific differences in the gene transcription.
Amino Acid Sequence↗
The human renin gene in transgenic mice.
We have obtained three transgenic mouse lines carrying the human renin gene. All three lines have been shown to transmit the introduced human gene to progeny. In the present study, we have demonstrated that the human renin gene was expressed and correctly spliced in the kidneys of both transgenic parents and progeny.
Animals↗
Expression of the human angiotensinogen gene in human cell lines.
The human angiotensinogen gene consists of five exons interrupted by four introns and spans 12 kilobases. The gene is expressed in liver and HepG2 cells derived from human hepatoma. To examine whether the angiotensinogen gene is expressed in extrahepatic cells, RNAs from kidney and several human cell lines have been isolated and analyzed by Northern blot hybridization with the cloned gene as a probe. The mRNA for angiotensinogen was detected in human kidney and human glioblastoma (A-172) cells. To investigate the endogenous regulation of angiotensinogen gene expression, HepG2 cells were cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase. The expression of the angiotensinogen gene was demonstrated to be completely suppressed by 3-aminobenzamide (10 mM).
Angiotensinogen↗
Tissue-specific expression of the human renin gene in transgenic mice.
Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5'-flanking sequence and 1.2 kb of 3'-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.
Animals↗
Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure.
We have isolated cDNA clones encoding boar acrosin, a serine protease participating in the initial stage of fertilization, from boar testis lambda gt11 cDNA libraries. Nucleotide sequencing of the overlapping clones indicates that the composite cDNA inserts contain 1,391 base pairs coding for a 5'-untranslated region, an open reading frame, a stop codon, a 3'-untranslated region, and a poly(A)+ tail. A polyadenylation signal, AATAAA, is located 33 bases upstream from the start of the poly(A)+ tail. The amino acid sequence deduced from the cDNAs shows that boar acrosin is initially synthesized as a prepro-protein with a 16-residue signal peptide at the NH2 terminus. This signal sequence is followed by a 399-residue sequence corresponding to the acrosin zymogen. COOH-terminal sequence analysis of boar sperm 55-kDa proacrosin and its processed forms indicates that the mature acrosin molecule contains 322 amino acid residues in two polypeptide chains, a 23-residue light chain and a 299-residue heavy chain, with a combined molecular mass of 35,735 Da, and that the 55-kDa proacrosin molecule has 14-, 18-, and 43-residue segments as COOH-terminal extensions that are removed during proacrosin maturation. The COOH-terminal 43-residue segment is rich in proline residues, including an unusual repeat of 23 consecutive prolines. The deduced amino acid sequence of boar acrosin shows a high degree of identity with major portions of other serine proteases, including the active site region and the location of cysteine residues. We conclude that boar acrosin is synthesized as a single-chain polypeptide with the regions corresponding to the light and heavy chains covalently connected by two disulfide bonds, and that the single-chain molecule is autoactivated by cleavage of the Arg23-Val24 bond after removal of the COOH-terminal 14-residue segment, resulting in the formation of the light and heavy chains. This two-chain molecule is then converted to the mature enzyme by removal of the COOH-terminal 18- and 43-residue segments.
Acrosin↗
Renin expression in the kidney and brain is reciprocally controlled by captopril.
The tissue distribution of rat renin mRNA was examined. Sensitive RNase protection analyses demonstrated that renin mRNA are produced by the extra-renal tissues such as adrenal, brain, liver, lung, pituitary and testis. In response to sodium depletion and captopril treatment, the expression of mRNAs encoding rat renin were in a tissue-specific manner. The level of kidney renin mRNA remarkably increased in sodium-depleted rats treated with captopril, whereas that of brain renin mRNA definitely decreased. No significant change in the level of liver renin mRNA was observed after the same treatment. These results suggest that the expression of cerebral renin is regulated by physiological stimuli independent of its extra-cerebral expression.
Animals↗
Ectopic production of renin by ileal carcinoma.
A 54-yr-old woman with the symptoms of primary reninism, i.e. hypertension, metabolic alkalosis and elevated levels of plasma renin activity (PRA) and aldosterone, is described. She had an ileal cancer secreting active and inactive renin. The symptoms markedly improved after resection of the tumor. In the tumor active and inactive renin were proved to be present by an assay of angiotensin I formation in the presence and absence of renin antibody, and renin immunoreactivity was found immunohistochemically. The mRNA coding for the renin precursor was identified in the RNA-rich extract of the tumor by blot hybridization analysis with the human renin cDNA as a probe. The mRNA from the tumor was shown to be identical in molecular size to that from the human kidney by agarose gel electrophoresis. This is the first description of an ectopic renin-producing ileal carcinoma and the first demonstration of renin mRNA in the tumor tissue.
Carcinoma, Small Cell↗
Structure of the rat renin gene.
We have isolated the renin gene from a Sprague-Dawley rat genomic library and determined its complete nucleotide sequence. The single rat renin gene is approximately 11,000 bases in length and consists of nine exons and eight introns. The amino acid sequence predicted from the genomic sequence indicates that the rat renin precursor consists of 402 amino acid residues, and shows 85%, 82% and 68% homology to the mouse Ren-1 and Ren-2, and human renins, respectively. The canonical promoter "TATA" boxes, TATAAA and TAATAA, are found 27 and 57 base-pairs upstream from the putative cap site, respectively. Several attractive regulatory sequences analogous to glucocorticoid, estrogen receptor-binding sites, cAMP-responsive element and SV40 enhancer core sequences were noted in the 5'-flanking region of the gene. In the first intron, segments with an average size of 38 base-pairs containing a NcoI site are present at 46 tandem repeats within 1710 base-pairs. A "CA" element consisting of (CA)27 was identified in intron 3. Furthermore, intron 8 contains a sequence that shows about 93% homology to that of the neuronal identifier sequence.
Amino Acid Sequence↗