Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The murine CEACAM9 and CEACAM11-related proteins as well as the pregnancy-specific glycoproteins (PSG) are secreted members of the CEA family which are differentially expressed in fetal trophoblast cell populations during placental development. PSG are essential for a successful pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. In contrast, Ceacam10 mRNA, coding for a protein identical in structure with CEACAM11-related proteins, is expressed in the maternal decidua surrounding the implantation site of the conceptus only during early stages of gestation between day 6.5 and day 10.5 postcoitum. To determine its role during murine development, we inactivated Ceacam10. Ceacam10(-/-) mice developed, like the previously established Ceacam9(-/-) mice, indistinguishably from wild-type littermates with respect to sex ratio, weight gain, and fertility. However, a small but significant reduction of the litter size by 23% was observed in Ceacam10(-/-) matings. Furthermore, combining the Ceacam9 and Ceacam10 null alleles, both located on chromosome 7, by meiotic recombination and subsequent mating of heterozygotes carrying both knockout alleles on one chromosome yielded wild-type and double knockout offspring at the expected Mendelian ratio. Taken together, both Ceacam10 and Ceacam9, alone or in combination, are not essential for either murine placental and embryonic development or for adult life.     

Norepinephrine-induced cardiac hypertrophy and fibrosis are not due to mast cell degranulation

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg·h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg·h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.     

Development of the nervous system in the "head" of <Emphasis Type="Italic">Limulus polyphemus</Emphasis> (Chelicerata: Xiphosura): morphological evidence for a correspondence between the segments of the chelicerae and of the (first) antennae of Mandibulata

We investigated brain development in the horseshoe crab Limulus polyphemus and several other arthropods via immunocytochemical methods, i.e. antibody stainings against acetylated alpha-tubulin and synapsin. According to the traditional view, the first appendage-bearing segment in chelicerates (the chelicerae) is not homologous to the first appendage-bearing segment of mandibulates (first antenna, deutocerebrum) but to the segment of the second antenna (tritocerebrum) or the intercalary segment in hexapods and myriapods. Accordingly, the segment of the deutocerebrum in chelicerates would be completely reduced. The main arguments for this view are: (1) the postoral origin of the cheliceral ganglion, (2) a poststomodaeal commissure, and (3) a connection of the cheliceral ganglion to the stomatogastric system. Our data show that these arguments are not convincing. During the development of horseshoe crabs there is no evidence for a former additional segment in front of the chelicerae. Instead, comparison of the brain structure (neuropil ring) between chelicerates, crustaceans and insects shows remarkable similarities. Furthermore, the cheliceral commissure in horseshoe crabs runs mainly praestomodaeal, which would be unique for a tritocerebral commissure. An unbiased view of the developing nervous system in the "head" of chelicerates, crustaceans and insects leads to a homologisation of the cheliceral segment and that of the (first) antenna (= deutocerebrum) of mandibulates that is also congruous to the interpretation of the Hox gene expression patterns. Thus, our data provide morphological evidence for the existence of a chelicerate deutocerebrum.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

Intracellular pathways and degradation of endosomal contents in basal epithelial cells of freshwater sponges (Porifera, Spongillidae)

 The digestive system expressed by basal epithelial cells of the freshwater sponges Spongilla lacustris and Ephydatia muelleri is mainly represented by a population of 30–50 preexisting lysosomes located in the close vicinity of the central nucleus. The strongly acidic vacuoles (pH 4–4.5) vary in size between 1 and 3 μm, and contain a set of different lysosomal enzymes. Immunocytochemical studies succeeded in the detection of β-hexosaminidase, cathepsin D, acid phosphatase, and α-glucosidase. Endosomes resulting from fluid-phase macropinocytosis, receptor-mediated endocytosis, or phagocytotic activity deliver their exogenous contents to the preexisting lysosomes for enzymatic degradation. Macropinosomes and phagosomes follow a rather reduced intracellular pathway by immediate fusion with the lysosomal compartment, whereas substances conveyed by coated vesicles pass through two additional vacuolar stages, namely early and late endosomes. Early endosomes serve as sorting organelles and segregate various constituents of complex ligands (BSA-AU_6, BSA-AU₁₂) by size into individual late endosomes, which then coalesce with preexisting lysosomes. As a whole, the intracellular pathways and hydrolytic processing of endosomal and phagosomal contents in freshwater sponge cells share certain similarities with the respective mechanisms in cells of higher eukaryotes. Accepted: 7 October 1997     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

Linking aphid honeydew, throughfall, and forest floor solution chemistry of Norway spruce

In a seminatural manipulation experiment with artificial irrigation we followed throughfall and forest floor solution chemistry collected underneath aphid infested and uninfested Norway spruce. Solutions underneath infested trees showed significantly higher concentrations of dissolved organic carbon (DOC) but lower concentrations of dissolved organic nitrogen (DON), NO₃-N, and NH₄-N in throughfall solutions and of NH₄-N in forest floor solutions. Average concentrations were 40.5% (DON), 27.5% (NO₃-N), and 46.2% lower (NH₄-N) underneath infested trees in throughfall solutions, and 19.5% (DON), 9.4% (NO₃-N), and 42.0% (NH₄-N) lower in forest floor solutions. Differences in throughfall were more pronounced than in forest floor leachates. It is likely that honeydew is fuelling the metabolism of micro-organisms and thus critically affects above and below ground nutrient cycles. We emphasize the importance of linking the biology of herbivores and micro-organisms with geochemical processes.     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.     

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background

Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.

Results

Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Conclusions

The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.