The possible role of metallothioneins in copper tolerance of Silene cucubalus

Growth and copper-binding of a copper-tolerant and a copper-sensitive population of Silene cucubalus (L.) Wib. have been studied. The copper-tolerant plants showed a much lower uptake and a proportionally higher transport of copper from root to shoot. A copper-binding protein with an apparent M_r of 8500 resembling metallothionein has been isolated from the roots of copper-treated plants of the tolerant population. After 20 d, the protein was observed to be inducible upon copper supply in the copper-tolerant plants, but not yet in the sensitive ones. This could be an indication of a difference in metalregulated synthesis of the protein. Ion-exchange chromatography of the 8500 protein yielded a major copper-containing fraction eluting at high ionic strength. Other characteristics such as UV absorption and amino-acid composition resembled strongly those of metallothioneins. The involvement of metallothioneins in the detoxification of copper within Cu-tolerant plants is discussed in relation to other mechanisms.Abbreviation DEAE diethyloaminoethyl     

Active pyrimidine absorption by chicken colon

Pyrimidine absorption by chicken large intestine was investigated employing the everted sac and flux chamber techniques. 3H-labelled uracil was used as substrate. The small intestine and the colon unlike the caecum, transported uracil from the mucosal to the serosal surface against a concentration gradient in the everted sac experiments. Furthermore, there was a net transport of uracil from the mucosal to the serosal side of the colon and jejunum in the flux chamber experiments. Uracil transport by the everted colon sacs against a concentration gradient was inhibited when the purine hypoxanthine was present in the incubation medium. Uracil transport by the everted colon sacs was also inhibited under anaerobic conditions and when 2,4-dinitrophenol was present in the incubation medium. Replacing the Na+ ions of the incubation medium by Li+ ions also caused an inhibition of uracil transport. It is concluded from these results that uracil (and probably other pyrimidines) are absorbed from the chicken colon by a Na+ ion-dependent active transport process having also an affinity for purines.     

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.     

Neurosecretion XVII. Experimentally induced release of neurosecretory material by exocytosis in the insect Leucophaea maderae

Summary In the corpora cardiaca of the insect Leucophaea the administration of serotonin elicits ultrastructural features indicative of the extrusion of neurosecretory material by exocytosis. The response to the stimulus and the process of extrusion seem to occur at considerable speed. Nearly all of the 30 test animals, fixed at various intervals starting as early as 3 min after the injection of the drug, show granules captured at the moment of leaving the axon as well as fully exteriorized secretory material. The fact that many of these granules are much smaller than the typical neurosecretory type speaks for intracellular fragmentation of the latter prior to the discharge of this cellular product. After 25 min or more the extruded electron dense structures show signs of breakdown. The apparent speed of these phenomena accounts for the dearth of omega-type configurations observed in unstimulated specimens of this species. The possible relationship between the membrane phenomena involved in exocytosis and the transient protrusions of bounding membranes of neurosecretory granules described in earlier papers remains to be clarified.Supported by N.S.F. research grant BMS 74-12456     

Effect of parental feeding activity on squab-induced crop sac growth in ring doves (Streptopelia risoria)

Effects of parental regurgitation feeding activity on crop sac development were studied in mate-separated male and female ring doves given 2 hr of daily exposure to food-deprived or recently fed squabs, for 4 consecutive days during the early posthatching period of the breeding cycle. In both sexes, food-deprived squabs stimulated more squab-directed activity, more parental regurgitation feeding activity, and greater crop sac development than recently fed young. Crop sac weights of males in both groups tended to be positively correlated with one or more parental activities. Correlations obtained in males exposed to food-deprived young further suggested that tactile stimuli associated with regurgitation behavior may promote crop sac development. In contrast to males, crop sac weights of females in both groups were not highly correlated with any type of contact-related parental activity or group of activities. These results, together with previous findings, suggest that nontactile stimuli from young played some role in mediating female crop sac weight differences in the two exposure conditions.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

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.