| 1. |
- Jia, X C, et al.
(författare)
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Expression of human luteinizing hormone (LH) receptor : interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species.
- 1991
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Ingår i: Molecular Endocrinology. - 0888-8809 .- 1944-9917. ; 5:6, s. 759-68
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Tidskriftsartikel (refereegranskat)abstract
- Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.
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| 2. |
- LaPolt, P S, et al.
(författare)
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Basic fibroblast growth factor induction of granulosa cell tissue-type plasminogen activator expression and oocyte maturation : potential role as a paracrine ovarian hormone.
- 1990
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 127:5, s. 2357-63
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Tidskriftsartikel (refereegranskat)abstract
- Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.
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| 3. |
- Feng, P, et al.
(författare)
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The structure of the TATA-less rat tissue-type plasminogen activator gene. Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:4, s. 2022-7
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Tidskriftsartikel (refereegranskat)abstract
- The genomic region carrying the rat tissue-type plasminogen activator (tPA) gene including its 5'-flanking sequence has been isolated and characterized by restriction enzyme analysis, Southern blotting, and DNA sequencing of all coding parts and the promoter region. The gene is approximately 25 kilobase pairs in size and comprises 14 exons separated by 13 introns. All the exon/intron boundaries agree with the GT-AG rule. The organization of the rat tPA gene is very similar to its human counterpart, and the location of the introns in the protein structure is identical to the human tPA gene. To characterize the promoter region, the transcription initiation site was identified by S1 nuclease protection experiments. A DNA fragment carrying 621 nucleotides of the 5'-flanking sequence was found to confer basal promoter activity and hormone responsiveness to a reporter gene construct in primary cultures of rat granulosa cells. Analysis of the rat tPA promoter sequence and a comparison with the human and mouse counterparts reveal several species-specific differences: the rat and mouse tPA promoters lack typical TATA and CAAT sequences found in the human tPA gene. Furthermore, the rat tPA promoter contains a consensus cAMP-responsive element shown to be required for cAMP responsiveness in eucaryotic genes. At the same position as the cAMP-responsive element in the rat gene, the mouse and human tPA genes have a 12-O-tetradecanoylphorbol-13-acetate-responsive element known to mediate activation by phorbol esters. The differences in the promoter sequences of the rat, mouse, and human tPA genes may have implications for the regulation of the tPA gene in different species.
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| 7. |
- Ohlsson, M, et al.
(författare)
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Transcriptional regulation of the rat tissue type plasminogen activator gene : localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.
- 1993
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 13:1, s. 266-75
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Tidskriftsartikel (refereegranskat)abstract
- We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.
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| 8. |
- Oikawa, M, et al.
(författare)
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Expression of gonadotropin-releasing hormone and prothymosin-alpha messenger ribonucleic acid in the ovary.
- 1990
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 127:5, s. 2350-6
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Tidskriftsartikel (refereegranskat)abstract
- GnRH exerts paradoxical effects on ovarian cells through specific receptors. Based on observed direct effects of GnRH and its antagonists on ovarian functions, the presence of endogenous ovarian GnRH-like peptide(s) has been postulated. In an attempt to detect the ovarian expression of GnRH or related genes at the RNA level, we used the reverse transcription-polymerase chain reaction (RT-PCR) to amplify GnRH mRNA levels. Total RNA from rat ovaries was converted to first strand cDNA using reverse transcriptase and amplified in PCR using a pair of primers complementary to the rat GnRH cDNA. The DNA products obtained were subcloned into plasmid vectors, and their sequences were determined. The most prominent PCR product of 462 basepairs (bp) was unexpectedly identified as a fragment of prothymosin-alpha cDNA previously found in the spleen. This cDNA was obtained because of an identical 10 bp match with the 3' end of one of the GnRH primers. Northern blot analyses using the cloned prothymosin-alpha cDNA as probe revealed the presence of mRNA for this factor in ovary, thymus, testis, placenta, and hypothalamus. RT-PCR amplification of hypothalamus and granulosa cell messages indicated the presence of a 244-bp product with a sequence identical to that of GnRH. To further confirm the presence of GnRH messages in the ovary, a second set of GnRH primers was used. PCR amplification of cDNA from hypothalamus, granulosa cells, and whole ovary yielded a 241-bp product identical to the authentic GnRH sequence based on analysis on both strands. In contrast, no PCR product was evident after amplification of thyroid cDNA. Our data demonstrated the expression of mRNA for GnRH and prothymosin-alpha in the ovary. Although the exact ovarian role of the immune hormone awaits further study, the detection of GnRH transcript in the ovary suggests potential intragonadal roles of this decapeptide.
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| 10. |
- Strandberg, L, et al.
(författare)
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Fluorescence studies on plasminogen activator inhibitor 1 : reactive centre cysteine mutants remain active after fluorophore attachment.
- 1994
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 76:3, s. 253-67
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Tidskriftsartikel (refereegranskat)abstract
- To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.
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