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K E Bernstein

Publications and source records attributed to K E Bernstein.

At least 73 records · Page 4Linked to original sources
PubMed1993

Regulated expression of testis angiotensin-converting enzyme during spermatogenesis in mice.

A unique isozyme of angiotensin-converting enzyme (ACE) is produced in large amounts by developing germ cells and is referred to as testis ACE. This protein results from the transcriptional recognition of a sperm-specific promoter during spermatogenesis. The expression within germ cells of testis ACE mRNA was studied using in situ hybridization, ribonuclease (RNase) protection, and Northern analysis. In parallel, testis ACE protein expression was measured through use of Western blot analysis and immunohistochemistry. Although testis ACE protein is first detected in step 10 spermatids, testis ACE mRNA is first detected in a developmentally younger cell population, late pachytene spermatocytes. Thus, testis ACE mRNA is translationally arrested for a period of several days until late in spermiogenesis.

Animals↗
PubMed1993

Sperm-specific expression of angiotensin-converting enzyme (ACE) is mediated by a 91-base-pair promoter containing a CRE-like element.

The gene encoding the testis isozyme of angiotensin-converting enzyme (testis ACE) is one example of the many genes expressed uniquely during spermatogenesis. This protein is expressed by developing germ cells late in their development and results from the activation of a sperm-specific promoter that is located within intron 12 of the gene encoding the somatic isozyme of ACE. In vitro transcription, DNase footprinting, gel shift assays, and transgenic mouse studies have been used to define the minimal testes ACE promoter and to characterize DNA-protein interactions mediating germ cell-specific expression. These studies show that proper cell- and stage-specific expression of testis ACE requires only a small portion of the immediate upstream sequence extending to -91. A critical motif within this core promoter is a cyclic AMP-responsive element sequence that interacts with a testis-specific transactivating factor. Since this putative cyclic AMP-responsive element has been conserved within the testis ACE promoters of different species and is found at the same site in other genes that are expressed specifically in the testis, it may provide a common mechanism for the recognition of sperm-specific promoters.

Animals↗
PubMed1993

Immunohistochemical localization of rat angiotensin II AT1 receptor.

To study receptors for angiotensin II, polyclonal rabbit anti-peptide antisera were prepared against the peptide QDDCPKAGRHC corresponding to amino acids 15-24 of the rat AT1A and AT1B receptors. Western analysis of rat tissues showed a major band of approximately 43 kDa. The antisera immunoprecipitated AT1-receptor protein produced in vitro. Immunohistochemical analysis of rat tissues showed intense staining of arterial and arteriolar smooth muscle. Other tissues that contained AT1-receptor protein included hepatocytes, the zona glomerulosa of the adrenal gland, and the smooth muscle of the bronchus, gut, ureter, and epididymis. In the kidney, intense staining was observed in all small arteries and arterioles. Both afferent and efferent arterioles contain approximately equal intensities of immunoreactive AT1 protein. The inner stripe of the outer medulla has a moderate level of receptors within thick ascending limb epithelium. Proximal tubular epithelium also expresses receptor protein. Glomerular immunoreactive AT1 protein is found within mesangial cells and varies in intensity among different rat strains. Lewis and Wistar rats demonstrated moderate glomerular staining, whereas the CD and Sprague-Dawley strains showed lesser levels of reactivity. The fact that glomerular mesangial cells are the primary locus of angiotensin II action within the glomerulus.

Amino Acid Sequence↗
PubMed1992

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

Angiotensin II↗
PubMed1992

The genomic organization of the rat AT1 angiotensin receptor.

The AT1 receptor subtype modulates all of the hemodynamic effects of the vasoactive peptide, angiotensin II. In this report, we investigate the genomic organization of this important receptor. A rat genomic library was screened with fragments from the 5' region of a previously cloned cDNA, pCa18b, encoding the rat AT1 receptor. Two lambda clones were isolated and the hybridizing restriction fragments were sequenced. Comparison of the genomic and cDNA sequences reveals that the rat AT1 receptor has three exons. Two of the exons encode 5' untranslated sequence while the third exon encompasses the entire coding region, a small portion of the 5' untranslated region and the entire 3' untranslated sequence. Further analysis of the genomic sequence 5' to the start site of pCa18b demonstrates typical sequence motifs found in many eukaryotic promoters including a TATA box, a cap site and a potential Sp1 binding site. Southern analysis of genomic DNA indicates that the AT1 receptor subtype represented by pCa18b is encoded by one gene within the rat genome.

Amino Acid Sequence↗
PubMed1992

Tissue specific expression of angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) is a component of the renin-angiotensin system and is critical in the homeostatic control of systemic blood pressure. There are two isozymes of ACE that result from two distinct promoter regions with the single ACE gene. In this article, we discuss the biochemistry of tissue specific promoter recognition as exemplified by the ACE gene.

Animals↗
PubMed1992

Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endothelial cell growth arrest.

Angiotensin converting enzyme (ACE) is a zinc-containing dipeptidase that converts angiotensin I to angiotensin II, a powerful vasoconstrictor and smooth muscle growth factor. ACE activity has been shown to be dynamically regulated by hormones, ACE inhibitors, and endothelial cell growth state. To study how ACE expression is regulated, we isolated and sequenced the bovine ACE gene using both ACE-specific cDNA and genomic clones. Bovine ACE cDNA encodes a single polypeptide of 1,306 residues with a molecular mass of 150 kd. Bovine ACE is approximately 80% homologous to that of other species. It contains two homologous domains of equal size. Alignment of ACE sequences from bovine, human, mouse, and rabbit reveals that during evolution both domains have been highly conserved. We used the bovine ACE cDNA to study regulation of ACE gene expression during density-dependent growth arrest. As endothelial cells became growth-arrested (6 days after confluence), there was a 12-fold increase in ACE activity and a 90% decrease in DNA synthesis. Immunocytochemically detectable ACE markedly increased in growth-arrested cells. The increase in ACE was due to increased ACE gene expression, as assayed by RNase protection, which showed a 20-fold increase in ACE-specific mRNA. The present study shows that bovine ACE is highly regulated by endothelial cell growth state at the level of protein and mRNA expression. Such dynamic regulation may have important consequences for angiotensin II production during endothelial cell proliferation after arterial injury.

Amino Acid Sequence↗
PubMed1991

Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.

There are two isozymes of angiotensin-converting enzyme (ACE), one produced by somatic tissues and a smaller protein synthesized by developing spermatozoa (testis ACE). To investigate the molecular control of testis ACE, we generated mice transgenic for a construct containing a putative testis-specific ACE promoter linked to the Escherichia coli reporter gene encoding beta-galactosidase. The transgenic mice express beta-galactosidase protein and RNA only within the testis. Histochemical analysis of the transgenic mice shows co-localization of beta-galactosidase protein and endogenous ACE within elongating spermatozoa. These studies demonstrate that transcription of testis ACE is controlled by a strong intragenic testis-specific promoter that is contained within a 698-base pair fragment immediately upstream from the transcription start site of testis ACE. Characterization of the testis ACE promoter may provide insights into the molecular mechanisms controlling cell stage-specific gene expression in the male germ line.

Animals↗
PubMed1991

Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor.

Angiotensin II is an important effector molecule controlling blood pressure and volume in the cardiovascular system. Its importance is manifested by the efficacy of angiotensin-converting enzyme inhibitors in the treatment of hypertension and congestive heart failure. Angiotensin II interacts with two pharmacologically distinct subtypes of cell-surface receptors, AT1 and AT2. AT1 receptors seem to mediate the major cardiovascular effects of angiotensin II. Here we report the isolation by expression cloning of a complementary DNA encoding a unique protein with the pharmacological specificity of a vascular AT1 receptor. Hydropathic modelling of the deduced protein suggests that it shares the seven-transmembrane-region motif with the G protein-coupled receptor superfamily. Knowledge of the AT1 receptor primary sequence should now permit structural analysis, definition of the angiotensin II receptor gene family and delineation of the contribution of AT receptors to the genetic component of hypertension.

Amino Acid Sequence↗
PubMed1991

Insulin-like growth factor I gene expression in vascular cells.

Insulin-like growth factor I (IGF I), a potent growth factor in vitro, is present in blood and in multiple tissues and is a major mediator of the effects of growth hormone on postnatal growth. IGF I is internalized and retained largely intact in cultured vascular endothelial cells. Neovasculature transiently expresses IGF I immunoreactivity, but it is not known whether this represents internalization of the circulating growth factor or vascular cell synthesis of IGF I. As an initial approach to defining the role of endogenous production of IGF I in the growth program of the vessel wall, Northern hybridizations were performed with RNA from cultured rat aortic smooth muscle cells and bovine aortic endothelial cells. Rat aortic smooth muscle cells expressed three primary IGF I messenger RNA transcripts sized 8.2, 1.7, and 0.9-1.2 kb. Bovine aortic endothelial cells expressed one major and one minor IGF I transcript of 2.1 and 1.6 kb, respectively. IGF I gene expression in smooth muscle cells was also demonstrated by ribonuclease protection assays using a rat exon 3 riboprobe. Both endothelial and vascular smooth muscle cells secreted IGF I, as detected by radioimmunoassay of conditioned medium after separation of IGF I from its binding proteins by gel filtration chromatography. Because IGF I stimulates growth of vascular cells, characterization of IGF I gene expression in blood vessels may be key to understanding developmental as well as abnormal growth in the cardiovascular system.

Animals↗
PubMed1990

Genomic DNA 5' to the mouse and human angiotensin-converting enzyme genes contains two distinct regions of conserved sequence.

Genomic DNA 5' to the mouse and human genes encoding angiotensin-converting enzyme has been isolated and analyzed. A sequence comparison identifies two discrete regions of genomic DNA that are highly conserved. One region, found immediately 5' to the mouse and human ACE genes, is a GC rich segment that contains a "TATA box" and several potential Spl binding sites. The second conserved region is found further 5' and contains several potential regulatory cis elements including a possible glucocorticoid responsive element. These two regions of genomic DNA may influence the rate of mRNA transcription from the angiotensin-converting enzyme gene.

Animals↗
PubMed1990

A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents.

Two biotinylated derivatives of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were synthesized. Compounds BL11 (epsilon-biotinamidocaproyl-lisinopril) and BL19 (epsilon-biotinamidocaproyl-beta-alanyl-beta-alanyl-lisinopril) have, respectively, 11 and 19 atoms of spacing structure between the biotin and the inhibitor moieties. Both compounds were found to be potent inhibitors of mouse kidney ACE, but they lost this ability in the presence of streptavidin in free solution. However, BL19 (but not BL11), when complexed to ACE, retained enough residual binding strength for streptavidin to allow the complex to be specifically removed from solution by streptavidin-agarose beads. It was thus possible to employ BL19 for the affinity isolation of ACE from crude mixtures. These results indicate that the bound determinant of lisinopril must lie at least 11 A below the outer surface of the ACE molecule.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1990

Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene.

Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptidyl carboxypeptidase that catalyzes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II. By analyzing cDNA and genomic DNA, we have constructed a consensus sequence encoding the testis isozyme of mouse ACE. Testis ACE cDNA contains 2,435 base pairs and encodes a protein of 732 amino acids. The N-terminal 66 amino acids are unique to the testis isozyme, while the remaining 666 are identical to the carboxyl half of mouse somatic ACE. The overall conservation of amino acid sequence between the testis isozymes of the mouse, rabbit, and human is 78 to 84%. The conservation of amino acids for the N-terminal domain uniquely expressed within the testis is 63 to 67% between these species. Primer extension and RNase protection experiments show that RNA transcription of the testis ACE isozyme begins 16 or 17 bases upstream from the translation start site. A sequence element resembling a TATA box is found 25 bases 5' of the transcription start site. To create its unique isozyme of ACE, the testis begins mRNA transcription in the middle of the exonic-intronic structure of somatic ACE, within a sequence treated as an intron by somatic tissues. Testis ACE is not the result of alternative RNA splicing but seems due to the start of transcription at a unique site within the ACE gene.

Amino Acid Sequence↗
PubMed1989

Mouse angiotensin-converting enzyme is a protein composed of two homologous domains.

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts angiotensin I into the potent vasoconstrictor angiotensin II. We have used cDNA and genomic sequences to assemble a composite cDNA, ACE.315, encoding the entire amino acid sequence of mouse converting enzyme. ACE.315 contains 4838 base pairs and encodes a protein of 1278 amino acids (147.4 kDa) after removal of a 34-amino acid signal peptide. Within the protein, there are two large areas of homologous sequence, each containing a potential Zn-binding region and catalytic site. These homologous regions are approximately half the size of the whole ACE protein and suggest that the modern ACE gene is the duplicated product of a precursor gene. Mouse ACE is 83% homologous to human ACE in both nucleic acid and amino acid sequence, and like human ACE, contains a hydrophobic region in the carboxyl terminus that probably anchors the enzyme to the cell membrane (Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G., and Corvol, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9386-9390). Northern analysis of mouse kidney, lung, and testis RNA demonstrates that the testicular isozyme of ACE is encoded by a single, smaller RNA (2500 bases) than the two message sizes found in kidney or lung (4900 and 4150 bases), and that this testicular RNA hybridizes to the 3' portion of ACE.315.

Amino Acid Sequence↗