Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

Induction of G protein-coupled estrogen receptor (GPER) and nuclear steroid hormone receptors by gonadotropins in human granulosa cells

Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation. The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH. Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized granulosa cells.     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

Ultrastructural and cytochemical examination of the nuclear crystalloid inclusions of Olea europaea L. mesophyll cells

Summary The nuclei of mesophyll cells of olive trees contain numerous sizeable crystalloid inclusions. Cytochemical examination using epoxy resin-embedded, semithin-sectioned tissue indicated the presence of proteins and oligoor polysaccharides in these inclusions. Their electron microscopical analysis revealed a crystalline substructure consisting of intersected subunits of high order. The spacing of the lattice fibrils and the angles of intersection were determined and used to establish a model of the unit cell of crystallization. It is suggested that the nuclear crystalloids of olive trees consist of glycoprotein molecules. They differ from the intranuclear crystalloids observed in other species predominantly in the high density of their subunit arrangement.     

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

With the aim to adapt high‐yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus‐induced apoptosis, and virus‐induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis‐related proteins, the earlier onset of virus‐induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza‐induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.     

Influence of various soil factors on the effects of ferulic acid on leaf expansion of cucumber seedlings

Summary Cucumber seedlings were grown in a Portsmouth soil-sand system to study how varying soil clay and organic matter content might modify cucumber seedling response to ferulic acid, a reported allelopathic agent. Leaf area expansion of cucumber seedlings, soil respiration, and soil solution concentrations of ferulic acid were monitored. Leaf area, mean absolute rates of leaf expansion, and shoot dry weight of cucumber seedlings were significantly reduced by ferulic acid concentrations ranging from 10 to 70 μg/g dry soil. Ferulic acid was applied every other day, since it rapidly disappeared from soil solution as a result of retention by soil particles, utilization by microbes and/or uptake by roots. The amount of ferulic acid retained (i.e., adsorbed, polymerized,etc.) by soil particles appeared to be secondary to microbial utilization and/or uptake by roots. Varying clay (5.3 to 9.8 g/cup) and organic matter (2.0 to 0.04g/cup) contents of the soil appeared to have little impact on the disappearance of ferulic acid from soil solution under “ideal” growth conditions for cucumber seedlings unless larger amounts of ferulic acid were added to the soil; in this case 200 μg/g. The addition of ferulic acid to the soil materials substantially increased the activity of the soil microbes. This latter conclusion is based on recovery of ferulic acid from soil solution and soil respiration measurements. Paper No. 10347 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N C 27695-7601. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named, nor criticism of similar ones not mentioned.