Structural simplicity of theZonula Occludens in the electrolyte secreting epithelium of the avian salt gland

Summary The structure of thezonula occludens in the secretory epithelium of the salt gland of the domestic duck was determined by thin section and freeze-fracture electron microscopy. These glands secrete an effluent with a NaCl concentration four times that of plasma, and thus maintain a steep ionic gradient across their secretory epithelium. Freezefracture replicas from salt stressed ducks demonstrate that thezonula occludens is surprisingly shallow in depth (20–25 nm) and generally consists of two parallel junctional strands which are juxaposed along their entire length. In addition to the simplicity of the junction separating mucosal and serosal compartments, the ratio of junctional length to apical surface area is large since luminal surfaces of secretory cells are narrow and intermesh with one another. Thezonula occludens in nonsecreting fresh water-adapted birds is similar to the salt stressed group except that two sets of double strand junctions are seen in addition to junctions consisting of a single set. Based on previous ultrastructural, cytochemical and physiological studies in salt glands and in other epithelia, a model for salt secretion was suggested in which intercellular space Na⁺, generated by basolateral ouabain-sensitive Na⁺ pumps, reaches the lumen via a paracellular route (Ernst & Mills, 1977,J. Cell Biol. 75:74). The simplicity of the morphological appearance of thezonula occludens in the salt gland, which resembles that described for several epithelia known to be leaky to ions, is consistent with this hypothesis.     

Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion.

We have used the human Lewis blood group fucosyltransferase cDNA and cross-hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1----4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x-negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM-1-dependent cell adhesion.     

Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.     

Granulocyte-macrophage colony-stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor-dependent hematopoietic cell line.

Steel factor (SF), the ligand for the proto-oncogene c-kit, acts synergistically with GM-CSF or IL-3 to support the growth of normal human hematopoietic progenitor cells. We examined the effects of SF on GM-CSF or IL-3 induced proliferation of a human factor-dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL-3 or GM-CSF alone, and its addition dramatically enhanced (three- to sixfold) maximal GM-CSF or IL-3 stimulated proliferation. SF did not increase the number or affinity of cell surface GM-CSF receptors. We examined several early events of signal transduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinase, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM-CSF and IL-3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140-150, 116, 106, 94, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal transduction intermediates known to be phosphorylated and activated by GM-CSF and IL-3, the 70-75 kDa Raf-1 kinase, and p42 mitogen-activated protein kinase-2 (MAPK), were also phosphorylated by SF. Combinations of GM-CSF or IL-3 with SF did not further increase the phosphorylation of Raf-1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM-CSF or IL-3, induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). These results indicate that SF and GM-CSF/IL-3 have partially overlapping effects on early signal transducing events, as well as striking differences, such as tyrosine phosphorylation of PLC-gamma. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.     

Cytokines involved in monocyte mediated tumor cell death and growth inhibition in serum-free medium.

In a serum-free culture system, the release of TNF, lI-1, lI-6, IFN-alpha, and IFN-beta during interaction of elutriated human monocytes (MO) with human tumor cells (TC) was studied by ELISA-technique. Contributions of these cytokines to inhibition of TC-growth and to induction of TC-death by supernatants (SU) gained from such MO/TC-interaction cultures were investigated using affinity chromatography for removal of individual cytokines. Although the TC used are relatively insensitive to recombinant human TNF, withdrawal of TNF causes 50% to 75% reduction of SU-induced TC-death rates, suggesting that susceptibility to TNF is raised during MO/TC-interaction by the other cytokines. Individual removal of other cytokines does not cause reduction of SU-mediated TC-death. However, combined withdrawal of lI-1 and IFN-alpha/beta causes in 2 of 4 TC-lines significant reduction of TC-death. Combined removal of TNF, IFN-alpha/beta, lI-1, and lI-6 leads to complete prevention of SU-mediated growth inhibitory and lytic effects, suggesting that besides these cytokines other signals are not involved significantly. SU-effects can be mimicked by appropriate combinations of authentic cytokines. The response of TC to SU- or cytokine-exposure is strikingly dependent on TC-density, leading at subconfluent TC-density exclusively to inhibition of growth and at postconfluent TC-density to induction of cell death. The principal effect of SU or cytokine combinations in this context seems to be the activation of growth inhibitory signal transduction pathways leading to TC-death in postconfluent TC-populations exclusively if growth stimulatory pathways are activated at the same time. Mouse L cells do not follow this reaction pattern: Their death is exclusively dependent on the presence of TNF in SU and they die upon SU-exposure at postconfluent as well as at subconfluent cell density.     

Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.

The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.