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1.
Molecular cloning and characterization of a progesterone-dependent cat endometrial secretory protein complementary deoxyribonucleic acid 总被引:1,自引:0,他引:1
R C Jaffe K M Donnelly P A Mavrogianis H G Verhage 《Molecular endocrinology (Baltimore, Md.)》1989,3(11):1807-1814
In the cat, a group of low molecular weight secretory proteins have previously been shown to appear in the endometrium after progesterone (P) administration to an estrogen (E2)-primed animal. Using a polyclonal antibody to these progesterone-dependent proteins (PDP) we have isolated a recombinant cDNA clone corresponding to the mRNA for PDP from a cDNA library prepared using poly(A+) RNA from the endometrium of P-treated E2-primed cats. Comparison of Western blots using the polyclonal antibody and epitope selected antibody demonstrated that the multiple molecular weight and isoelectric forms of the PDP are immunologically related and potentially products of the same gene. Northern analysis revealed that the mRNA for the PDP in the endometrium of P-treated E2-primed cats was 1.8 kilobases in length. Using slot blot analysis, we found that the PDP mRNA levels were low in the endometrium of ovariectomized animals and undetectable in E2-treated animals. With 1 day of P treatment the PDP mRNA levels were readily detectable and they peaked after 5 to 7 days of P treatment. No PDP mRNA was detectable in myometrium, oviduct, or ovary. Sequence analysis revealed that PDP had significant homology to human, rat, and mouse cathepsin L at the nucleotide (80%, 74%, and 73%, respectively) and amino acid (68%, 65%, and 63%, respectively) level. We suggest that PDP via its collagenolytic and elastolytic activities as a cathepsin L is responsible for preparing the endometrium for blastocyst implantation. 相似文献
2.
The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Immunologiocal and biochemical methods were employed to demonstrate the presence of progestagen-dependent proteins in human endometrium. Cytosols were prepared from proliferative and secretory phase endometria of cycling women, from decidua and decidua-rich tissues of women in early pregnancy and from decidua of tubal pregnancy. Antisera were raised in rabbits against the antigens of decidua of tubal pregnancy and decidua-rich tissues. Immunoelectrophoresis, Ouchterlony's immunodiffusion test and polyacrylamide gel electrophoresis using native gels revealed 2 antigenic proteins, designated antigens A and B, in secretory endometria, decidua-rich tissues, decidua, and in decidua of tubal pregnancy. However, only 1 antigenic protein was detected by SDS-gel electrophoresis: antigens A and B may therefore be two different proteins or two forms of a single protein. The antigens could not be detected in non-pregnancy sera or in term placentae. Double isotopic labelling (incubation of tissues with [3H]- and [14C]leucine) followed by protein fractionation methods were used to compare the in-vitro synthesis rates of antigens in poroliferative tissues with those in decidua or secretory endometria. The rate of synthesis of antigens A and B was markedly higher in the decidua and secretory endometria than in the proliferative endometria. We conclude, therefore, that during progestagen-dependent transformation of proliferative phase endometria into secretory endometria and decidua in women, there is a selective stimulation of at least one species of pregnancy-associated protein. 相似文献
4.
This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P. 相似文献
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The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)
An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells. 相似文献
7.
DeSouza L Diehl G Yang EC Guo J Rodrigues MJ Romaschin AD Colgan TJ Siu KW 《Proteomics》2005,5(1):270-281
Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with similar conclusions. 相似文献
8.
In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process. 相似文献
9.
Secretory Protein I (SP-I) is an acidic glycoprotein that is stored and co-secreted with parathormone by parathyroid glands. It has been found to be chemically similar, if not identical, to chromogranin A of the adrenal medulla and to be present in most endocrine cells. In the present study, 35SO4 was shown to be incorporated into SP-I and several other proteins of porcine parathyroid tissue incubated in vitro. The predominant sulfated species secreted to the medium was SP-I. Up to 20% of the tyrosine residues in secreted SP-I were labeled with 35SO4. Both the cellular and secreted forms migrated on sodium dodecyl sulfate gels as a pair of proteins with apparent molecular weights of 82,000 and 78,000. The 82-kDa protein could be converted to the 78-kDa species by treatment with neuraminidase. Sulfate exists in SP-I as tyrosine sulfate based on the identification of this amino acid by thin layer electrophoresis following alkaline hydrolysis. Extracellular Ca2+ (3 mM) greatly suppressed the secretion of 35SO4-labeled SP-I without affecting the intracellular sulfation of the molecule or the secretion of a minor sulfated protein unrelated to SP-I. The ratio of incorporated 35SO4 to 3H-amino-acid was greater in secreted SP-I than in tissue SP-I, suggesting that much sulfation of this protein occurred during or just before secretion. 相似文献
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N Santoro D T MacLaughlin H H Bauer A A Hargraves H L Dichek G S Richardson 《Biology of reproduction》1989,40(5):1047-1055
Protein secretion by the human endometrium was studied in vitro in medium after incubation of tissue minces (n = 10) or glands isolated by collagenase digestion (n = 4) from tissues obtained at the time of curettage from normal women. Samples were incubated in a serum-free medium for 24 h at 38 degrees C in the presence of radiolabeled methionine. Dialyzed medium from each sample was subjected to two-dimensional gel separation, and detected by protein staining. Although 5 of the 27 proteins were considered to be present in the labeling experiments by only one of the three observers, there was agreement about the presence of the 22 others. In addition, the observers categorized the proteins into three groups for purposes of analysis: a) those associated with the follicular phase of the cycle; b) those associated with the luteal phase; and c) those not cycle-related. One protein triplet, labeled #27, showed a significant relation to the luteal phase (p less than 0.01). A complete lack of similarity between the pattern of labeled proteins obtained from the medium and labeled proteins obtained from lysates of cells incubated in the same experiments makes it unlikely that cellular lysis, as opposed to secretion, contributed to the pattern of proteins studied in these experiments. 相似文献
12.
Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar 总被引:1,自引:0,他引:1
We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development. 相似文献
13.
Steveson TC Zhao GC Keutmann HT Mains RE Eipper BA 《The Journal of biological chemistry》2001,276(43):40326-40337
Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential for the biosynthesis of amidated peptides, was used to assess the role of cytosolic acidic clusters in trafficking to regulated secretory granules. Casein kinase II phosphorylates Ser(949) and Thr(946) of PAM, generating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identified by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific for phospho-Ser(949)-PAM-CD demonstrates that a small fraction of the PAM-1 localized to the perinuclear region bears this modification. Pituitary cell lines expressing PAM-1 mutants that mimic (TS/DD) or prevent (TS/AA) phosphorylation at these sites were studied. PAM-1 TS/AA yields a lumenal monooxygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granules more efficiently than wild-type PAM-1 and monooxygenase release is more responsive to secretagogue. Furthermore, this acidic cluster affects exit of internalized PAM-antibody complexes from late endosomes; internalized PAM-1 TS/DD accumulates in a late endocytic compartment instead of the trans-Golgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking. 相似文献
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15.
The recycling of a secretory granule membrane protein 总被引:2,自引:0,他引:2
We have used N-hydroxysuccinimido-d-biotin as a reagent for labeling proteins exposed at the surface of cultured bovine adrenal chromaffin cells during Ba2+-stimulated secretion. A specific secretory granule membrane constituent, dopamine-beta-hydroxylase (DBH), has been investigated using immunoprecipitation followed by electrophoresis. Within 30 min of stimulation, exposed DBH had been cleared from the cell surface. Nevertheless, quantitation of labeled DBH using [125I] streptavidin suggested that it remained undegraded over a period of 24 h, a time during which secretory granule stores of catecholamines were being replenished. Subcellular fractionation of the cultured cells suggested that, after 3 or 4 h, the biotinylated DBH, which was still membrane-bound, was located in particulate material that also contained cytochrome b561, another major secretory granule membrane component. 相似文献
16.
The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy. 相似文献
17.
A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h. 相似文献
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Kuroda A Nishimura T Ishida T Hirota R Nomura K 《Biotechnology and bioengineering》2008,99(2):285-289
In the current studies, we found that the DksA protein from Escherichia coli binds strongly to chrysotile, which is the most commonly used form of asbestos. We developed a convenient colorimetric assay for chrysotile using a fusion of DksA and alkaline phosphatase along with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium as substrates. Also, using a fusion of DksA and green-fluorescent protein, we were able to detect chrysotile by fluorescence microscopy. 相似文献
20.
The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone. 相似文献