Biomedical subjects

D Belin

Publications and source records attributed to D Belin.

At least 73 records · Page 4Linked to original sources

PubMed1989

Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2.

Two forms of plasminogen activators inhibitor 2 (PAI-2) are synthesized by human and murine monocytes/macrophages: one accumulates in the cytosol, while the other is translocated into the endoplasmic reticulum, glycosylated and secreted. We show here that a single mRNA encodes both forms of PAI-2. Firstly, a single PIA-2 mRNA was detected by Northern blot hybridization and by RNase protection. Secondly, transfection of a PAI-2 cDNA led to the synthesis of both forms of PAI-2. Finally, in vitro translation of an mRNA transcript of the PAI-2 cDNA in the presence of microsomal membranes generated two topologically distinct forms of PAI-2. The cytosolic and secreted forms of PAI-2 do not result from the use of two translation start sites, since their synthesis initiates at the same AUG, in a sequence context that is conserved between the human and murine genes. Thus, the accumulation of one polypeptide into two topologically distinct cellular compartments can be achieved by facultative translocation.

Amino Acid Sequence↗

PubMed1989

Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos.

To assess in vivo the postulated participation of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in processes involving tissue remodeling and cell migration, we have studied the cellular distribution of u-PA and t-PA mRNAs during mouse oogenesis and embryo implantation. By in situ hybridizations, we detected t-PA mRNA in oocytes and u-PA mRNA in granulosa and thecal cells from preovulatory follicles. These findings are compatible with a role for plasminogen activators in oogenesis and follicular disruption. We demonstrated the presence of u-PA mRNA in the invasive and migrating trophoblast cells of 5.5- and 6.5-d-old embryos. At 7.5 days, u-PA mRNA was predominantly localized to trophoblast cells that had reached the deep layers of the uterine wall, while the peripheral trophoblast cells surrounding the presomite stage embryo were devoid of specific signal. In 8.5-d-old embryos abundant u-PA mRNA expression resumed transiently in the giant trophoblast cells at the periphery of the embryo and in the trophoblast cells of the ectoplacental cone, to become undetectable in 10.5-d-old embryos. These observations establish the in vivo expression of the u-PA gene by invading and migrating trophoblast cells in a biphasic time pattern; they are in agreement with the proposed involvement of the enzyme in the extracellular proteolysis accompanying embryo implantation.

Animals↗

PubMed1989

Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes.

The translational activation of dormant tissue-type plasminogen activator mRNA during meiotic maturation of mouse oocytes is accompanied by elongation of its 3'-poly(A) tract. Injected RNA fragments that correspond to part of the 3'-untranslated region (3'UTR) of this mRNA are also subject to regulated polyadenylation. Chimeric mRNAs containing part of this 3'UTR are polyadenylated and translated following resumption of meiosis. Polyadenylation and translation of chimeric mRNAs require both specific sequences in the 3'UTR and the canonical 3'-processing signal AAUAAA. Injection of 3'-blocked mRNAs and in vitro polyadenylated mRNAs shows that the presence of a long poly(A) tract is necessary and sufficient for translation. These results establish a role for regulated polyadenylation in the post-transcriptional control of gene expression.

Animals↗

PubMed1989

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium.

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.

Animals↗

PubMed1989

Receptor-mediated phagocytosis by macrophages induces a calcium-dependent transient increase in c-fos transcription.

The transcription of the c-fos gene and the level of c-fos mRNA in mouse peritoneal macrophages are rapidly, strongly and transiently increased after Fc- and C3b-mediated phagocytosis, but not after phagocytosis of latex particles. In order to induce both phagocytosis and a rise in c-fos mRNA, binding to receptors must be followed by mobilization of Ca++ from intracellular Induction of c-fos transcription in macrophages by other agents acting through different intracellular "messengers', i.e. phorbol esters (protein kinase C), cholera toxin (cAMP) and dexamethasone (glucocorticoid receptor) also depends on intracellular Ca++. In all these conditions, induction of c-fos transcription is inhibited by the calmodulin antagonist W7, suggesting a common Ca++-dependent pathway for c-fos gene activation in macrophages.

Animals↗

PubMed1988

A rabbit Ig lambda L chain C region gene encoding C21 allotopes.

Southern blot analyses of germ-line DNA obtained from rabbits expressing lambda chains of C7 and/or C21 allotypes were performed with a rabbit C lambda region-specific probe; a 12-kbp EcoRI- and a 2-kbp BamHI-hybridizing fragment were detected only in the DNA from rabbits expressing the C21 allotype. The 12-kbp EcoRI fragment was cloned and shown to contain two C lambda region-encoding genes in the same orientation. Each is preceded by a J lambda gene segment. Nonamer-12-bp spacer-heptamer recombination signal sequences were found 5' of each J lambda segment, and splicing signals were identified at the 3' ends of the J lambda segments and the 5' ends of the corresponding C lambda genes. The C lambda 5 gene, which exhibits a sequence identical with that found in several cDNA clones, is carried by the 2-kbp BamHI fragment missing from the genomic DNA of rabbits which do not express the C21 allotype. The second C lambda gene, C lambda 6, lies 3' of C lambda 5, in a 1.6-kbp BamHI fragment which is present in genomic DNAs of all tested rabbits, irrespective of their phenotype. Its sequence is identical with that found in one cDNA clone and differs from that of C lambda 5 in 17 base positions resulting in four amino acid substitutions. A fragment of a cDNA, with a J-C region sequence identical with that encoded by the J lambda 5-C lambda 5 gene pair, was subcloned into a plasmid expression vector. The resulting polypeptide product could be specifically immunoprecipitated by anti-C21 but not anti-C7 alloantisera, showing that some, if not all, C21 allotopes are encoded by the C lambda 5 gene. In contrast, the C lambda 6 gene product was not precipitable, either by anti-C7 or by anti-C21 alloantisera, although it was readily immunoprecipitated by a goat anti-rabbit lambda chain antiserum.

Amino Acid Sequence↗

PubMed1988

Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes.

Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.

Animals↗

PubMed1988

Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E.

Gene 32 from bacteriophage T4 is transcribed as precursor transcripts which are processed to a stable product. This processing of the gene 32 mRNA was observed in RNase III or P-deficient strains of Escherichia coli. However, after infection of an RNase E-deficient strain, the amount of processed transcript was significantly reduced while the levels of the precursor transcripts remained high. RNase E therefore appears to have an essential role in the processing of the gene 32 mRNA. We have mapped the exact 5' end of the processed transcript by primer extension. The cleavage occurs near a stem-loop structure at a site which shows some similarity to other known RNase E cleavage sites. The effects of the processing on the differential stability of the upstream and downstream sequences, and on gene expression, are discussed.

Autoradiography↗

PubMed1988

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases.

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.

Animals↗

PubMed1987

Plasminogen activator-specific inhibitors in mouse macrophages: in vivo and in vitro modulation of their synthesis and secretion.

Mouse resident peritoneal macrophages synthesize two plasminogen activator-specific inhibitors (PAI) that are functionally and antigenically related, but differ in their apparent Mr and oligosaccharide content. Most of the Mr 40,000 inhibitor can be recovered from the cell lysate, whereas the Mr 55,000 glycosylated PAI is preferentially secreted. The murine macrophage PAI are functionally similar and immunologically related to PAI synthesized and secreted by human monocytes-macrophages, and to a PAI from human placenta (PAI-2). PAI production by murine mononuclear phagocytes can be modulated both in vivo and in vitro. Bone marrow-derived macrophages do not produce detectable PAI, whereas inflammatory macrophages obtained from thioglycollate-induced peritoneal exudates produce only low levels of PAI. In cultures of resident peritoneal macrophages, phorbol myristate acetate and cholera toxin increase the synthesis of the Mr 55,000 secreted PAI, whereas dexamethasone decreases the synthesis of both PAI; the production of PAI is also enhanced in the presence of macrophage colony-stimulating factor (CSF-1). The overall proteolytic activity of mononuclear phagocytes thus depends in part on the controlled synthesis and secretion of PAI. The balance between the production of plasminogen activators and of their inhibitors could be critical in determining the level of plasminogen-dependent extracellular proteolysis associated with different phases of the inflammatory response.

Animals↗

PubMed1987

Increase of urokinase-type plasminogen activator gene expression in human lung and breast carcinomas.

To assess the postulated correlation between plasminogen activators (PAs) and malignancy, we determined the mRNA content for urokinase-type (u-PA) and tissue-type (t-PA) enzymes in a prospective series of 29 primary lung and 27 primary breast carcinomas. Dot blots of total RNAs were hybridized with appropriate cRNA probes under conditions that allow quantitative measurement of the mRNA level for each PA. Most tumors (43 of 56) had a u-PA mRNA content higher than the mean + 1 SD of nonmalignant tissue counterparts. A large, 4- to 20-fold, increase in u-PA mRNA content was demonstrated in 14 of 29 lung carcinomas and in 10 of 27 breast carcinomas. A statistically significant correlation (Fisher's test, P = 0.007) was found between elevated u-PA mRNA content in lung carcinomas and the presence of regional lymph node metastases. These results are consistent with a role for u-PA in tumor invasiveness and metastatic propensity and may have important prognostic and therapeutic implications.

Adenocarcinoma↗

PubMed1987

Modulations of functional activity in differentiated macrophages are accompanied by early and transient increase or decrease in c-fos gene transcription.

Marked changes in c-fos proto-oncogene mRNA level and transcription rate were observed upon modulation of the functional activity of cultured mouse peritoneal macrophages. Cholera toxin (CT), dexamethasone (dex), interferon-gamma (IFN-gamma), concanavalin A (Con A), and endotoxin (LPS) induced changes in mRNA levels and transcription rates of both urokinase-type plasminogen activator and tumor necrosis factor/cachectin genes, the products of which are sensitive indices of macrophage activity. All of these agents also caused rapid and transient changes in c-fos gene expression, either enhancement (CT, dex, and LPS) or decrease (IFN-gamma and Con A). Moreover, inhibition of protein synthesis elicited a transient increase in the level of c-fos gene transcription, suggesting that the transcriptional activity of the c-fos gene is controlled by labile protein repressor(s). Taken together, these results suggest a possible role for the c-fos gene product, a nuclear protein, in the modulation of the functional activity of differentiated macrophages.

Animals↗

PubMed1987

Amiloride selectively inhibits the urokinase-type plasminogen activator.

The diuretic drug amiloride, an inhibitor of Na+ uptake, competitively inhibits the catalytic activity of the urokinase-type plasminogen activator (u-PA), with a Ki of 7 X 10(-6) M. Generation of plasmin, cleavage of peptide substrates, and interaction of u-PA with a specific macromolecular proteinase inhibitor are all prevented in the presence of the drug. In contrast, amiloride does not affect the activity of either tissue-type plasminogen activator, plasmin, plasma kallikrein or thrombin. The inhibition of u-PA by amiloride may be related to the previously reported inhibition of u-PA-type enzymes by Na+. Amiloride or related compounds could prove useful in selectively controlling u-PA-catalyzed extracellular proteolysis.

Amiloride↗

PubMed1987

Sense and antisense transcription of bacteriophage T4 gene 32. Processing and stability of the mRNAs.

Analysis of bacteriophage T4 gene 32 transcription has revealed a multiplicity of mRNAs. In plasmids, gene 32 is expressed primarily from a strong promoter that is shut off after phage infection. In a wild-type infection, gene 32 is initially transcribed from prereplicative polycistronic and monocistronic promoters; subsequently, a monocistronic late mRNA predominates. This transcript, as well as a post-transcriptionally processed product of the earlier mRNA, can be stable. The eventual degradation of the stable mRNAs is temporally regulated by the phage. Finally, the transcription termination region of gene 32 can function as an antisense promoter both in vitro and in vivo.

Base Sequence↗

PubMed1987

Plasminogen activator-specific inhibitors produced by human monocytes/macrophages.

Human monocytes/macrophages produce plasminogen activator-specific inhibitors (PAIs) that form covalent complexes with urokinase-type plasminogen activator (uPA). We have characterized two functionally and antigenically related forms of PAIs produced by resting and phorbol myristate acetate (PMA)-treated U 937 cells: an Mr 40,000 form, presumably nonglycosylated, with a pI of 5.2, that is constitutively synthetized by these cells and that remains predominantly intracellular; a PMA-induced form of heterogeneous Mr (50,000-65,000) with a pI of 4.7, that is preferentially secreted; this PAI is glycosylated with terminal sialic acid residue(s). Biosynthetic labeling experiments demonstrated that both PAIs are synthetized by U 937 cells. They are inactivated upon treatment with propanol, heat, and acid; the covalent and equimolar complexes formed between these PAIs and 125I-uPA are dissociated by ammonium hydroxide, suggesting that the PAIs are linked to uPA via an ester bond. Human peripheral blood monocytes/macrophages also produce the two forms of PAI. These PAIs are clearly different from the main plasma protease inhibitors and they are both antigenically related to the PAI-2 characterized in human placenta.

Cell Line↗

PubMed1987

Glucocorticoid modulation of plasminogen activators and of one of their inhibitors in the human mammary carcinoma cell line MDA-MB-231.

In cultures of the human mammary carcinoma-derived cell line MDA-MB-231, plasminogen activator (PA) activity was reduced substantially following treatment with the glucocorticoid dexamethasone. These cells produced urokinase-type PA (u-PA) and tissue-type PA (t-PA), and both enzymes were decreased in dexamethasone-treated cultures. The drop in u-PA activity was associated with a decrease in the synthesis of single-chain pro-u-PA and in the concentration of u-PA messenger RNA; however, the decrease in u-PA activity was more extensive than could be accounted for by inhibition of enzyme synthesis only, suggesting that postsynthetic events were also involved. The comparatively small dexamethasone-induced decrease in t-PA activity was not associated with a change in the concentration of t-PA messenger RNA. Hence, the two PA genes are differentially regulated by the same hormone. MDA-MB-231 cells also produced a PA-specific inhibitor related to that produced by bovine aortic endothelial cells (PAI-1). This inhibitor was present in two forms: one functionally active, and the other which required activation by sodium dodecyl sulfate; both forms were increased in cultures exposed to dexamethasone. Thus, glucocorticoid-induced inhibition of PA activity in these cells results from a decrease in u-PA synthesis and a concomitant increase in the production of a PA inhibitor.

Breast Neoplasms↗

PubMed1987

Chromosomal assignments of genes for tissue plasminogen activator and urokinase in mouse.

The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.

Animals↗

PubMed1987

Antagonist effect of RU 486 on transcription of glucocorticoid-regulated genes.

The effect of RU 486, a synthetic steroid that is a powerful antagonist of glucocorticoid hormones, was tested on the transcription of several glucocorticoid-regulated genes in different cell types: inflammatory murine macrophages and two human mammary gland-derived cell lines, MDA-MB-231 and HBL-100. The transcription of genes which are positively regulated by glucocorticoids (e.g., tissue-type plasminogen activator and c-myc in mammary cells, c-fos in macrophages) and that of genes which are negatively regulated by these agents (e.g., urokinase-type plasminogen activator in all three cell types, TNF-a and IL-1 in macrophages) was explored. RU 486 almost completely prevented the effects of dexamethasone on the transcription of these various genes. When added alone, RU 486 had essentially no agonist activity.

Animals↗