Collagen and elastin gene expression in the hypertensive pulmonary artery of the rat.
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Animals↗
Biomedical subjects
J Rosenbloom
Publications and source records attributed to J Rosenbloom.
Structure of the 3' portion of the bovine elastin gene.
A bovine genomic library constructed by partial Sau3A digestion and contained in lambda Charon 30 was screened by in situ hybridization with a 1.3-kilobase (kb) sheep elastin cDNA clone [Yoon, K., May, M., Goldstein, N., Indik, Z., Oliver, L., Boyd, C., & Rosenbloom, J. (1984) Biochem. Biophys. Res. Commun. 118, 261-269]. Three clones encompassing 10 kb of the bovine elastin gene were identified and characterized by restriction mapping and DNA sequencing of the 6.2 kb of the most 3' region of the gene. These analyses have permitted localization of eight exons in the 6.2 kb in which the translated exons vary in size from 27 to 69 base pairs, and there is an approximately 1-kb untranslated region at the 3' end. In addition to identification of sequences homologous to those found in porcine tropoelastin, the analyses defined a 58 amino acid sequence that forms the carboxy-terminal region of tropoelastin, and this sequence, which contains two cysteine residues, was previously not observed in the protein sequence data. The analyses also suggest that functionally distinct cross-link and hydrophobic domains of the protein are encoded in separate exons.
Amino Acid Sequence↗
Selective inhibition of excessive scleroderma fibroblast collagen production by lymphokines from normal human mononuclear cells.
We studied the effects of lymphokines from mitogen-stimulated normal human mononuclear cells on collagen production by confluent monolayer cultures of scleroderma dermal fibroblasts. We examined 10 cell lines obtained from patients with early onset, rapidly progressive disease. All cell lines exhibited increased collagen production, compared with cell lines from age- and sex-matched normal individuals. We found that supernatants from phytohemagglutinin-stimulated mononuclear cells caused greater than 45% inhibition of collagen production by the cultured scleroderma fibroblasts; the degree of inhibition was concentration-dependent. Partially purified lymphokine preparations produced 84-94% inhibition of collagen production by scleroderma cells. The lymphokine effects were shown to be selective for collagen, since the synthesis of noncollagenous proteins was not significantly affected. The results suggest that lymphokines from normal mononuclear cells can modulate the excessive collagen biosynthesis that is characteristic of scleroderma fibroblasts, and these lymphokines may have a place as possible therapeutic agents for this incurable disease.
Adult↗
Monoclonal antibodies to different epitopes in amelogenins from fetal bovine teeth recognize high-molecular-weight components.
Enamel proteins were extracted and partitioned into amelogenin and enamelin fractions. Although several attempts were made to raise monoclonal antibodies to each protein fraction, monoclonal antibodies were only obtained against the amelogenin fraction. Six monoclonal antibodies were generated, and these could be classified into three groups recognizing different epitopes by a competitive enzyme-linked immuno-absorbent assay. A model for the arrangement of the epitopes is proposed. In Western-blotting experiments, all six monoclonal antibodies recognized amelogenin components of approx. 45,000 and 60,000 daltons as well as lower molecular-weight components of 10,000 to 30,000. It is proposed that the 45,000 and 60,000 dalton components are precursors of the lower molecular-weight components.
Amelogenin↗
Production of collagen synthesis inhibitory lymphokine by human leukemic T-lymphocyte cell lines.
It has been previously shown that normal human peripheral blood mononuclear cells produce a soluble factor(s) that causes selective inhibition of collagen production by cultured human diploid fibroblasts, and that stimulation of the mononuclear cells by appropriate mitogens greatly enhances its production. Because of the difficulties inherent in obtaining sufficient peripheral blood lymphocytes to purify the collagen synthesis inhibitory factor (CSIF) further and to study in detail its mechanisms of action, we have examined CSIF production by several leukemic human T- and B-lymphocyte cell lines. From the 10 lines tested (4 T- and 6 B-lymphocyte cell lines), we found 2 T-lymphocyte cell lines which produced CSIF constitutively. When the effects of various lymphocyte mitogens on CSIF production by these cell lines were examined, it was found that only phorbol myristic acid was stimulatory. Further studies demonstrated that optimal CSIF production by the phorbol myristic acid-stimulated leukemic T-lymphocytes could be achieved in a chemically defined, serum-free medium. The CSIF in the supernatants from such cultures was further purified by ammonium sulfate precipitation and gel filtration chromatography and it was shown that it had similar properties to those of CSIF produced by normal human peripheral blood lymphocytes. These findings indicate that certain leukemic T-lymphocyte cell lines are capable of CSIF production in continuous cultures. These results will permit the large scale production of CSIF and a better understanding of its mechanisms of action.
B-Lymphocytes↗
Restricted homology between human alpha 1 type IV and other procollagen chains.
Screening of a human fibroblast cDNA library with a mouse type IV procollagen clone resulted in one 1.05-kilobase isolate that was used to identify a 1.7-kilobase clone overlapping the former by less than 150 nucleotides. EcoRII digestion revealed that the larger clone exhibited the pattern characteristic of DNA coding for a collagenous sequence. Blot hybridization to RNA from mouse F9 stem cells and from these cells treated with retinoic acid and N6, O2'-dibutyryl-cAMP showed induction of type IV mRNA. DNA sequencing and comparison of the derived amino acids with the reported protein data demonstrated that the clones encode part of the alpha 1 chain of human type IV procollagen. Alignment of alpha 1 (IV) with other human procollagens showed minimal but detectable homology. A small cluster of charged residues in the alpha chain is partially shared by type IV. In close proximity is an interruption in the alpha 1 (IV) Gly-Xaa-Yaa region corresponding to the 3' end of a unique proline-free, and therefore also less rigid, area in other collagen triple helices. Analysis of the carboxyl-terminal alpha 1 (IV) peptide showed a repeat symmetry possibly dictated by the six cysteines in each half of the structure. The position of five cysteines in addition to four tyrosine/tryptophan groups allowed a correlation to be drawn between the 3' noncollagenous type IV region and the larger, highly conserved carboxyl propeptides of other human procollagens. Such similarities in the different chains may define functional domains conserved throughout evolution.
Animals↗
Utilization of a peroxidase antiperoxidase complex in an enzyme-linked immunosorbent assay of elastin-derived peptides in human plasma.
Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and significant impairment of lung function usually occurs before the disease is diagnosed. Because lung elastin degradation appears to be a prerequisite for the development of the disease, immunologic detection of elastin-derived peptides in the blood might be an effective approach to the early detection and monitoring of the disease. We here report an improved enzyme-linked immunosorbent assay for elastin peptides using a peroxidase-antiperoxidase complex as the reporter group. The assay is sensitive to 2 ng/ml elastin peptides. We show that for optimal, reproducible results the assay should be carried out at 16 degrees C rather than at room temperature and that determinations should be made on plasma containing protease inhibitors rather than on serum. The levels of elastin-derived peptides appeared to remain relatively constant when multiple samples were taken during a 5- to 10-wk period from individual subjects. In addition, patients with COPD had elevated elastin peptide levels (127 +/- 47 ng/ml) compared with levels in normal nonsmokers (58 +/- 17 ng/ml), whereas normal smokers had values intermediate between the 2 groups (mean peptide levels of 76 +/- 42 ng/ml). A small group of normal smokers (20%) had elevated elastin peptide levels similar to those in the emphysema group and may represent that group of smokers who are at risk of developing obstructive lung disease.
Animals↗
Chromosomal localization of the human elastin gene.
mRNA isolated from fetal human aorta was used to synthesize cDNA that was cloned into the PstI site of pBR322. The recombinant clones were screened with an authentic sheep elastin cDNA, and one human clone that hybridized strongly was isolated and characterized. The 421-base pair (bp) insert of this human clone was sequenced by the dideoxy method, and the DNA sequence showed strong homology to the nontranslated portion of the sheep elastin cDNA. This result unequivocally identified the human clone, designated pcHEL1, as an elastin clone. Plasmid pcHEL1 labeled with [3H] nucleotides was used in in situ hybridization experiments utilizing normal metaphase chromosomes and also with cells carrying a balanced translocation between chromosomes 1 and 2: 46,XY,t(1;2)(p36;q31). The results strongly suggest that the elastin gene is localized to the q31----qter region of chromosome 2.
Animals↗
Molecular cloning and carboxyl-propeptide analysis of human type III procollagen.
Two human cDNA libraries prepared from normal fibroblast (GM3348) and rhabdomyosarcoma (CCL136) mRNAs were screened under cross hybridization conditions with a genomic fragment coding for exons 2 and 3 of the avian type III procollagen COOH-propeptide (Yamada, Y., Mudryj, M., Sullivan, M. and deCrombrugghe, B. (1983) J. Biol. Chem. 258, 2758-2761). One cDNA clone containing a 1.12 kb insert was isolated from the CCL136 library and later used to identify a GM3348 derived clone with a 2.4 kb insert. Comparison of the human and avian type III C-terminal propeptides revealed much more divergence in the first 54 amino acids following the terminal cysteine of the triple helical region than is present in the alpha 1(I) and alpha 2(I) procollagen chains of these species. Analysis of poly (A+) RNA from normal human fibroblast and tumor cell lines showed that they differed greatly in the relative amounts of alpha 1(I), alpha 2(I), and alpha 1(III) mRNAs. Furthermore, as we previously reported for the alpha 1(I) and alpha 2(I) transcripts, multiple mRNAs also hybridize to the cloned alpha 1(III) DNA.
Amino Acid Sequence↗
Transcriptional control of human diploid fibroblast collagen synthesis by gamma-interferon.
Recombinant gamma-interferon (rec gamma-IFN) caused potent inhibition of collagen synthesis by cultured confluent human diploid fibroblasts in a dose-dependent manner. Gel electrophoresis of the newly synthesized proteins from the culture media of rec gamma-IFN-treated fibroblasts demonstrated a selective depression of procollagen without a significant change in non-collagenous proteins. Dot blot hybridization to a Type I procollagen cDNA probe showed that the inhibition of collagen production was accompanied by a decrease in the levels of collagen mRNA. These results indicate that rec gamma-IFN is capable of exerting transcriptional modulation of collagen biosynthesis and suggest that it may play an important role in regulation of normal and pathologic fibrogenesis.
Cells, Cultured↗
Characterization of a sheep elastin cDNA clone containing translated sequences.
mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.
Amino Acid Sequence↗
Secretion of elastin in the embryonic chick aorta as visualized by immunoelectron microscopy.
Recently, significant advances have been made in characterizing the pathway of elastin biosynthesis from the biochemical point of view and a 70,000 dalton protein, designated tropoelastin, appears to be the primary translation product and soluble intermediate of the insoluble elastin. However, relatively little is known concerning the intracellular secretory pathway of tropoelastin. We previously developed an electron microscopic technique using elastin-specific antibody and ferritin-conjugated secondary antibody to identify intracellular elastin and to identify, provisionally, intracellular vesicles containing elastin ( Damiano et al., Conn. Tiss . Res. 8: 185-188, 1981). However, the method did not permit localization of elastin in other intracellular organelles. We now describe an improved post-embedding technique using the peroxidase-antiperoxidase method to detect the primary elastin antibody and have localized elastin in both the endothelial and medial cells of the embryonic chick aorta. Specific staining was visualized in the cisternae of the endoplasmic reticulum, in the Golgi apparatus, and in vesicles forming on the trans side of the Golgi. Some of these smaller vesicles appeared to fuse, forming larger vesicles which may have a storage function. Both types of vesicles were seen fusing with the cell plasma membrane, suggesting that elastin is secreted by an exocytotic process. These results suggest that tropoelastin follows the classical pathway for protein secretion.
Animals↗
Selective inhibition of human diploid fibroblast collagen synthesis by interferons.
The effects of alpha- and gamma-interferons (IFNs) on collagen production by confluent human diploid fibroblasts in culture were examined. It was found that partially purified alpha-IFNs and affinity purified gamma-IFNs caused greater than 50% inhibition of collagen synthesis by these cells independently of their effect on cell proliferation. Recombinant alpha-IFNs showed a similar effect (38.8% inhibition), indicating that collagen synthesis inhibition was a constitutive property of IFNs. Collagen synthesis inhibition by IFNs was concentration dependent. Gel filtration chromatography of the newly synthesized proteins from the media of fibroblasts incubated with partially purified alpha-IFNs demonstrated a selective depression of molecules eluting in the region of procollagen. No detectable increase in collagen degradation products or underhydroxylation of procollagen was observed. Short-term kinetic studies further demonstrated that the major effect of IFNs was due to a net decrease in fibroblast collagen production rather than to impairment of secretion or increased extracellular degradation of the newly synthesized molecules. These results indicate that alpha- and gamma-IFNs are potent inhibitors of human fibroblast collagen production and suggest that they may play an important role in the regulation of normal and pathologic fibrogenesis.
Carbon Radioisotopes↗
Elastin: relation of protein and gene structure to disease.
The elastic properties of many tissues such as the lung, dermis, and large blood vessels are due to the presence of elastic fibers in the extracellular space. These fibers have been shown by biochemical and ultrastructural analysis to be comprised of two distinct components, a more abundant amorphous component and the microfibrillar component. The microfibrillar component is found in 10- to 12-nm fibrils which are located primarily around the periphery of the amorphous component but, to some extent, interspersed within it. The protein, elastin, makes up the highly insoluble amorphous component and is responsible for the elastic properties. Elastin is found throughout the vertebrate kingdom except for very primitive fish and possesses an unusual chemical composition consonant with its characteristic physical properties. Elastin is composed largely of glycine, proline, and other hydrophobic residues and contains multiple lysine-derived cross-links, such as the desmosines, which link the individual polypeptide chains into a rubber-like network. The intervening, hydrophobic regions of the polypeptide chains between the cross-links are highly mobile, and the elastic properties of the fibers can be described in terms of the theory of rubber elasticity. Recent application of recombinant DNA techniques has led to further understanding of the structure of elastin. Analyses of the bovine and human elastin genes have demonstrated that the hydrophobic and cross-linking domains are encoded in separate exons. These exons tend to be small, varying from 27 to 114 base pairs, and are separated by large intervening sequences. Furthermore, DNA sequence analysis has demonstrated that the elastin molecule contains two cysteine residues which were not previously identified near the carboxy terminus and which may be important in the interaction of elastin with other extracellular matrix proteins. Further DNA sequencing should determine the complete amino acid sequence of elastin. Biosynthetic studies and in vitro translation of elastin mRNA have demonstrated that a 72,000-dalton polypeptide, designated tropoelastin, is the initial translation product. Analysis of several developing systems has demonstrated that elastin synthesis is controlled by the level of elastin mRNA. After packaging into membrane-bound vesicles in the Golgi apparatus, tropoelastin is secreted by exocytosis into the extracellular space where it is cross-linked by a copper-requiring extracellular enzyme, lysyl oxidase. Elastin can be solubilized only by proteases that have consequently been designated elastases, although these are general, powerful proteases that can hydrolyze numerous proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis.
Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.
Chromatography, Gel↗
Immunologic measurement of elastin-derived peptides in human serum.
Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and usually significant impairment of lung function has occurred before the disease is diagnosed. It is likely that destruction of the elastic fiber is a prerequisite for the development of the disease, and it is possible that immunologic identification in the serum of peptides derived from lung elastin degradation might be an effective approach to the early detection and monitoring of the disease. We prepared antibodies to peptides derived from human lung parenchymal elastin and used these antibodies in an enzyme-linked immunosorbant assay to quantitate elastin-derived peptides in the serum of 39 normal control nonsmokers, 33 smokers with normal lung function, and 40 patients with COPD. On average, statistically significant higher levels of elastin-derived peptides were found in the normal smokers and COPD patients compared to the controls. Further work with larger numbers of subjects is necessary to determine whether such a test is effective in identifying those individuals who are at risk of developing COPD.
Antibodies↗
Immune modulation of connective tissue functions: studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells.
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Cell Adhesion↗
Analysis of elastin gene expression in the developing chick aorta using cloned elastin cDNA.
A segment of chick elastin cDNA cloned in the plasmid pBR322 was sequenced by the method of Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564) and the 192-base pair insert was found to be derived from the nontranslated region of the 3' end of the mRNA. The nick-translated cDNA was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single species of 3.5 kilobases hybridized to the cDNA probe and this species increased greatly between day 7 and day 14 of embryonic development. This increase was paralleled by an increase in translatable aortic elastin mRNA and by the in vivo rate of elastin synthesis, demonstrating that the change in synthesis during development is largely controlled by the elastin mRNA content of the aorta.
Animals↗