Sialic acid binding protein of human endometrium: Its regulation by steroids

In the present study, we have observed that sialic acid binding protein (SABP – a 54 kDa glycoprotein which was isolated from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females and purified to apparent homogeneity and was earlier reported from this laboratory) was found in sufficiently detectable amount in the endometrium of normal cycling women whereas it was found in lesser amount in tissue from women who have recently entered the post-menopause stage. SABP was observed in both follicular and luteal phase of menstrual cycle which was found by western blot analysis. In the de-novo synthesis experiment, synthesis and secretion of SABP was found to be stimulated by estradiol (E₂) whereas progesterone (P₄) was found to have no significant stimulatory effect on it which was also confirmed by HEC cell culture. In the HEC cell culture, priming of cells with E₂ was found to influence the effect of P₄ on SABP when it was added 2 h after E₂ administration. This was observed by doing immunoprecipitation followed by SDS-PAGE and autoradiography. Hence this report clearly indicates that E₂ regulates the synthesis and secretion of 54 kDa SABP from human endometrium. How E₂ priming of endometrium influences the effect of P₄ on SABP has been discussed.     

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.     

Possible interrelationship between vitamins E and b12 in the disturbance in methylmalonate metabolism in vitamin E deficiency.

The presence of three major proteins alpha, beta and gamma in rat ventral prostate was demonstrated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Their regulation by androgens was studied by measuring the rates of synthesis of the proteins in minced prostatic tissue by using L-[35S]methionine. The three proteins account for 30-40% of the proteins synthesized in the gland. After castration, their rates of synthesis rapidly decline to about 1% that of normal animals, and this cannot be accounted for by the accompanying decrease in general protein synthesis. Testosterone reverses these changes in castrated animals, so that after 4 days normal synthesis is restored. The regulation is specific for androgens, since cyproterone acetate, an anti-androgen, is inhibitory and oestradiol-17beta and corticosterone are without effect. Preliminary characterization of the proteins indicates that protein alpha (mol.wt. 22000, pI unknown) is a glycoprotein containing glucose and/or mannose residues and occurs in both the mitochondrial and cytosol fractions. Protein beta (mol.wt. 12000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also found exclusively in the cytosol fraction.     

Identification of immunodominant Trypanosoma musculi antigens recognized by monoclonal antibody and curative immunoglobulin G2a antibody.

Trypanosoma musculi obtained from normal or irradiated (900 rad) hosts or from in vitro cultures were lysed and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar protein banding patterns with a molecular weight (mol. wt) range from 34 to 68 kDa were observed between the two bloodstream forms. In comparison, lysates of cultured parasites showed a unique banding pattern of antigens within the same mol. wt range. Western blot of bloodstream form lysates, probed with immune plasma (IP), revealed a wide range of parasite proteins. However, when probed with the IgG2a-enriched fraction of IP, a major band of approximately 66 kDa was detected on the blot. Several bands of higher mol. wt were also observed. When anti-T. musculi monoclonal antibodies were used to probe the blot, the 66 kDa protein was again recognized. Using indirect fluorescence, live bloodstream form parasites were analysed by flow cytometry and the p66 protein was determined to be a surface molecule. Finally, lysates of 35S-methionine-labelled trypanosomes were immunoprecipitated with Sepharose linked anti-T. musculi monoclonal antibodies and the eluted ligand analysed by SDS-PAGE and autoradiographed. The 66 kDa band was identified, therefore confirming that this protein was of parasite origin.     

Liproprotein inhibitor of bone marrow cells in tumor-bearing rats.

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

LIPOPROTEIN INHIBITOR OF BQNE MARROW CELLS IN TUMOR-BEARING RATS

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. ⁵⁹Fe-heme synthesis, [³H]thymidine DNA synthesis, and ¹⁴C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (>400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipo-protein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Bidirectional interaction between unfolded-protein-response key protein HSPA5 and estrogen signaling in human endometrium

The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E(2)) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E(2) did not affect HSPA5 expression after 8-24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E(2) (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E(2) up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E(2) levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings.     

Platelet-activating factor stimulates phospholipase C activity in human endometrium.

Human preimplantation embryos secrete platelet-activating factor (PAF), which stimulates prostaglandin E2 synthesis from secretory endometrium. This study investigated the action of PAF on phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-specific phospholipase C activity in human endometrium. Slices of normal endometrium were incubated with 5 microCi/ml myo-[2-3H] inositol for 3 h at 37 degrees C in 95% O2 and 5% CO2 to label tissue phosphoinositides. Inositol phosphates were extracted using trichloroacetic acid precipitation and diethylether neutralization and production was measured using Dowex 1-X8 anion-exchange column chromatography. PAF induced rapid and concentration-dependent accumulation of inositol phosphates (IP) from secretory endometrium, but had no effect on endometrium removed in the proliferative phase of the menstrual cycle. The IP3 fraction was significantly elevated from a median value of 14.0 c.p.m. mg-1 dry wt [range: 8-41 c.p.m. mg-1 dry wt] to 28.0 c.p.m. mg-1 dry wt [range: 11-87 c.p.m. mg-1 dry wt, P less than 0.002] following 1 min exposure of secretory endometrium to PAF-acether, in the presence of 10 mM LiCl. PAF-induced hydrolysis of PtdIns(4,5)P2 was inhibited by the specific PAF receptor antagonist WEB 2086, in a dose-dependent manner (P less than 0.02), indicating that in human endometrium PtdIns(4,5)P2 hydrolysis is mediated via a PAF receptor. These results indicate that PAF receptor coupling activates endometrial PtdIns(4,5)P2-specific phospholipase C only in the secretory phase of the menstrual cycle, suggesting that the PAF response may be under ovarian steroid regulation. It is proposed that the ability of the endometrium to respond to PAF appears to be a feature of the preparation of this tissue for implantation and that the second messengers generated may play a role in cellular processes involved in the maternal recognition of very early human pregnancy.     

Soluble interferon-alpha receptor molecules are present in body fluids.

Soluble forms of the interferon-alpha receptor (sIFN-alpha R) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alpha R, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alpha R and [125I]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alpha R were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-alpha R prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be as high as 70,000 Da. It is therefore concluded that sIFN-alpha R molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alpha R, are present in human serum and in normal human urine.     

Protein synthesis and secretion by the human endometrium during the menstrual cycle and the effect of progesterone in vitro

To identify markers of endometrial differentiation specimens of endometrium from the menstrual cycle were incubated in vitro with [35S]methionine, in the absence or presence of progesterone, and protein synthesis and secretion were studied by fluorographic analysis of one dimensional SDS/gradient polyacrylamide gels. Changes were demonstrated in the rate of synthesis and secretion of a number of endometrial proteins (EP) during the cycle and in response to progesterone. Endometrial proteins were classified into three groups: Group I-synthesized and secreted throughout the menstrual cycle and unaffected by progesterone exposure; Group II-synthesis and secretion associated with histological type of endometrium and unaffected by progesterone exposure, e.g. EP 13 (Mr 33,000) with proliferative, EP 15 (Mr 28,000) with secretory and EP 14 (Mr 32,000) with late secretory endometrium; Group III-synthesis and secretion regulated by progesterone exposure irrespective of source of endometrium, e.g. EP 9 (Mr 54,000) and 11 (Mr 45,000). The Group II proteins EP 14 and 15 were also the major secretory protein products of endometrium from first and second trimester pregnancy respectively, the native forms referred to as pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG). We conclude that EP 15 (alpha 2-PEG) represents a human analogue of uteroglobin.     

Rapid purification of two lipocortin-like proteins from mice lung

We have purified two proteins (40 kDa and 36 kDa) from mice lung by the method of calcium-precipitation/EGTA solubilization and then a separation on a high anion exchanger column (Mono Q HR 5/5. Pharmacia) with a gradient of NaCl. The two proteins were strong inhibitors of phospholipase A2 as assessed in vitro with porcine pancreatic phospholipase A2 and [3H]-oleic acid labeled E. Coli membranes as substrate. The 40 kDa protein had a pI of 5.8 and was found to be immunologically related to human recombinant lipocortin I. The 36 kDa protein had a pI of 4.7 and cross-reacted with a polyclonal antibody raised against a 32 kDa human lipocortin-like protein described in human blood mononuclear cells. We report here a rapid purification of two distinct lipocortin-like proteins from mice lung.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

Monoclonal antibody against a nuclear matrix antigen in proliferating human cells

A monoclonal antibody of the IgG2a subclass was isolated from the supernate of a hybridoma line obtained with splenocytes from a mouse immunized with a crude nucleolar fraction of human Namalwa cells. This antibody identifies a single nuclear polypeptide antigen characterized by: (a) presence in proliferating human cell lines and phytohemagglutinin-stimulated lymphocytes, but absence in resting lymphocytes; (b) appearance in stimulated lymphocytes in parallel with the onset of DNA synthesis; (c) a speckled distribution in the nucleoplasm; (d) tight association with nuclear matrix structures identified by both biochemical and in situ extraction and enzyme treatment procedures; (e) mol wt of 125 kDa and pI 6.5 as determined by immunoprecipitation or immunoblotting of nuclear or nuclear matrix proteins fractionated by gel electrophoresis. The above characteristics identify the p125/6.5 nuclear matrix protein recognized by the isolated monoclonal antibody as belonging to the class of proliferating cell nuclear antigens.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Synthesis and release of polypeptides by the baboon (Papio anubis) uterine endometrium in culture

This study was designed to identify proteins released in culture by the baboon uterine endometrium. Endometrial tissues from cyclic baboons were minced and cultured in the presence of L-[3H]leucine or L-[35S]methionine for 24 h. The culture media and solubilized tissues were analyzed by one- and two-dimensional gel electrophoresis for secretory products that were uterine-specific. The fluorographs of the one- and two-dimensional gels demonstrated that the proteins released into culture media could be divided into two groups. Group I proteins were present throughout the menstrual cycle and showed minor cyclic variations in intensity, and Group II proteins were those that appeared to be hormonally modulated. Group I was comprised of several high molecular weight proteins (Mr greater than 200,000) and at least five additional proteins ranging in molecular weight from 80,000 to 37,000, with isoelectric points (pIs) of 5.1 to 6.0. Group II consisted of a protein (Mr 33,000; pI 7.6) that was observed only during the follicular stages of the cycle and two other groups of proteins (Mr 130,000 and 88,000) that were present during the luteal stage. Western blots of tissue culture media incubated with antibodies against human placental proteins (PP) and prolactin demonstrated that PP4 and PP7 were secreted throughout the cycle while PP12, PP16, and prolactin were only present during the luteal stage of the cycle. Thus, it appears that the baboon uterine endometrium, like that of the human, secretes a wide array of proteins in culture. Our results also suggest that a few of these proteins are immunologically similar. Endometrial differentiation during the menstrual cycle altered the secretion of some proteins, whereas the synthesis of others appeared to be dependent on either estrogen or progesterone and were stage-specific.     

Two-dimensional immunoblot analysis of antigenic proteins of Spirometra plerocercoid recognized by human patient sera

Sparganosis is caused by invasion of Spirometra plerocercoid into various tissues/organs. Subcutaneous sparganosis can be diagnosed and treated by worm removal, while visceral/cerebral sparganosis is not easy to diagnose. The diagnosis depends largely on the detection of specific antibodies circulating in the patients' sera. Previous studies demonstrated that 31 and 36kDa proteins of the sparganum invoked specific and sensitive antibody responses, but also showed cross reactions with cysticercosis sera. We enriched protein fractions containing 31-36kDa through gel filtration and examined immune recognition pattern against the patient sera by 2-dimensional electrophoresis (2-DE) followed by immunoblotting. Serum samples from sparganosis patients recognized 8-10 protein spots of 31 and 36kDa with different isoelectric point (pI) values with variable combinations, in which four spots of 31kDa with pIs 3.4, 3.9, 4.0 and 4.1, and one 36kDa spot (pI 3.5) appeared to be specifically reactive. One 31kDa protein spot with pI 3.3 and two spots of 36kDa with pIs 3.3 and 3.5 reacted crossly with neurocysticercosis sera. Neither sera from patients with other parasitic infections nor those from healthy controls showed positive reaction. Two-DE/immunoblot analysis might be highly available in differential serodiagnosis of human sparganosis.     

Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle

The human uterine epithelium is characterised by remarkable plasticity with cyclic changes in differentiation that are controlled by ovarian steroid hormones to optimise conditions for embryo implantation. To understand whether and how cell-cell adhesion is affected, the localisation of junction proteins was studied throughout the menstrual cycle. Expression patterns were examined by immunofluorescence in 36 human endometrial specimens of different cycle stages. Antibodies against the desmosomal proteins desmoplakin 1/2 (Dp 1/2) and desmoglein 2 (Dsg 2), the adherens junction proteins E-cadherin and β-catenin and also the common junctional linker protein plakoglobin showed a strong subapical staining during the proliferative phase until the early luteal phase (day 20). In the mid- to late luteal phase, however, these junctional proteins redistributed over the entire lateral plasma membranes. In contrast, tight junction proteins (ZO-1, claudin 4) remained at their characteristic subapical position throughout the menstrual cycle. mRNA levels of Dp 1/2, E-cadherin and ZO-1 obtained by real time RT-PCR were not significantly changed during the menstrual cycle. The observed redistribution of desmosomes and adherens junctions coincides with the onset of the so called implantation window of human endometrium. We propose that this change is controlled by ovarian steroids and prepares the endometrium for successful trophoblast invasion.     

Evidence for new cellular proteins which may negatively control DNA replication or cell growth in rat 3T3 fibroblasts

When separated and proliferating rat 3T3 cells are treated with butyrate (6 mM), DNA synthesis stops within 24 h, while RNA and protein synthesis proceed unaffected. This gradually converts normal cells into giant ones in the presence of butyrate (volume up to 30-fold greater). The giant cells stop growing when cell to cell contact is established. By studying the rate of synthesis of 300 cell proteins, we have identified two proteins (39 kDa, PI = 6.2, and 60 kDa, pI = 5.6) whose synthesis rises at least 10-fold when DNA replication and mitosis are prevented following intercellular contact or butyrate treatment, and another (64 kDa, pI = 5.6) whose synthesis rises at least 10-fold when cell growth stops by contact, both in the presence of butyrate and in the absence of butyrate (untreated confluent cells). The synthesis of some cellular oncogenes increases when the cell transits from G0 to S phase; the two proteins of 39 and 60 kDa described here are regulated in the opposite direction, their synthesis is enhanced when the cell leaves the proliferation cycle to enter G0.     

Progesterone induces a secretory protein in cultured rabbit endometrial stromal cells

Secretion of newly synthesized proteins by rabbit endometrial stromal cells in culture was studied. Progesterone (P) stimulated the synthesis and secretion of a protein with a mol. wt of approx 62 K and a pI of 6.5-7.0. Induction of 62 K protein synthesis was dose dependent; addition of 10 nM P to primary cultures caused a 2-fold or greater increase in the amount of this protein in the medium while addition of 1 microM P resulted in a 4.3-fold increase. Synthesis of this 62 K protein was not induced by estradiol, dexamethasone, or testosterone, nor was progesterone stimulation of the protein modified by estradiol. Thus, induction appears to be specific for a progesterone receptor mediated effect. Synthesis and secretion of this protein in the culture system described is potentially useful as a progestin bioassay.