Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells.

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double-immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.

Observation of extremely long spin relaxation times in an organic nanowire spin valve.

Organic semiconductors that are pi-conjugated are emerging as an important platform for 'spintronics', which purports to harness the spin degree of freedom of a charge carrier to store, process and/or communicate information. Here, we report the study of an organic nanowire spin valve device, 50 nm in diameter, consisting of a trilayer of ferromagnetic cobalt, an organic, Alq3, and ferromagnetic nickel. The measured spin relaxation time in the organic is found to be exceptionally long-between a few milliseconds and a second-and it is relatively temperature independent up to 100 K. Our experimental observations strongly suggest that the primary spin relaxation mechanism in the organic is the Elliott-Yafet mode, in which the spin relaxes whenever a carrier scatters and its velocity changes.

Biosynthesis of glycosylated human lysozyme mutants.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.

Synthesis and localization of myeloperoxidase protein in transfected BHK cells.

Processing and localization of myeloperoxidase was studied in nonmyeloid cells. For this purpose BHK cells were transfected with human myeloperoxidase cDNA. In the transfected cells a protein with mol wt of 85,000 was found, which reacted with the specific anti-human myeloperoxidase antiserum. In size and in sensitivity to endo-beta-N-acetylglucosaminidase H this protein resembled the myeloperoxidase precursor synthesized in human promyelocytes. Unlike in the promyelocytes, in BHK cells the 85,000-Da protein was not converted to 60,000- and 14,000-Da polypeptides of the mature enzyme. In Percoll gradients the protein was found predominantly in the light membrane fractions. Microscopic examination revealed a conspicuous immune reaction over the endoplasmic reticulum and nuclear membranes and a moderate labeling over lysosome-like organelles. Pulse-chase experiments indicated that the protein was slowly released from the endoplasmic reticulum; after 1 day the protein was found in similar amounts in cells and in the medium. The secreted protein contained at least one endo-beta-N-acetylglucosaminidase-resistant oligosaccharide. It is suggested that normal intracellular segregation of myeloperoxidase depends on a signal or component, which is not or incompletely expressed in BHK cells.

Lysosomal segregation of a mannose-rich glycoprotein imparted by the prosequence of myeloperoxidase.

The role of the N-terminal sequence of myeloperoxidase in the intracellular targeting was examined by using glycosylated lysozyme as a reporter. A fusion protein was constructed in which the presequence residues-18 through -6 of the lysozyme moiety had been replaced by residues 1-158 of prepromyeloperoxidase. Expression of the fusion protein in Chinese hamster ovary cells demonstrated its partial secretion and partial intracellular retention. The latter was accompanied by trimming the myeloperoxidase prosequence off the lysozyme moiety. The rate of the retention of the lysozyme fusion protein was higher than that of glycosylated lysozyme that had been expressed in cells transfected with cDNA of glycosylated lysozyme. The retention was insensitive to NH4Cl. In the secreted protein, lysozyme contained predominantly complex oligosaccharides as demonstrated by a proteolytic fragmentation in vitro and resistance to endo-beta-N-acetylglucosaminidase H. In contrast, when targeted to lysosomes, the lysozyme moiety of the fusion protein contained predominantly mannose-rich oligosaccharides. In baby hamster kidney cells, the trimming of the oligosaccharides in the lysozyme fragment was less vigorous, and a selective targeting of molecules bearing mannose-rich oligosaccharides to lysosomes was more apparent than in Chinese hamster ovary cells. In the presence of monensin, the formation of complex oligosaccharides in the fusion protein and its secretion were strongly inhibited, whereas the intracellular fragmentation was not. We suggest that the prosequence of myeloperoxidase participates in the intracellular routing of the precursor and that this routing operates on precursors bearing mannose-rich rather than terminally glycosylated oligosaccharides and diverts them from the secretory pathway at a site proximal to the monensin-sensitive compartment of the Golgi apparatus.

Interferon-gamma, mitomycin C, and cycloheximide as regulatory agents of MHC class II-associated invariant chain expression.

The murine and human major histocompatibility complex class II-associated invariant chain genes are expressed in mature B cells and in antigen-presenting cells. Several pre-B cell lines and fibroblasts do not naturally contain invariant chain mRNA. Expression is inducible, however, by interferons and other agents interfering with proliferation. Mitomycin C induces the transcription of the gene in pre-B cells, but not in fibroblasts. Interferon-gamma acts in both types of cells. Cycloheximide inhibits the induction of the invariant chain mRNA by interferon-gamma, suggesting that protein synthesis is required. In fact, cycloheximide itself increases the transcriptional rate at the invariant chain gene, suggesting the existence of a labile repressor or an indirect action through cycloheximide arrest of the cell cycle. Lipopolysaccharide (LPS) activation of B lymphocytes causes a rapid decrease of the invariant chain mRNA level and of the amount of invariant chain protein due to rapid turnover. Also class II alpha and beta mRNA expression decreases after LPS treatment. The decrease of invariant chain protein is accompanied by increased surface expression of alpha and beta. The murine invariant chain gene transfected into human fibroblasts is regulated by the same agents and the same dose of agents as is the endogenous gene. The differentiation marker invariant chain thus seems to be transcribed from a gene that is accessible to regulation even in nonlymphoid cells and the expression of which is linked to states of nonproliferation. The sequence responsible for these responses is contained within the cloned genomic fragment and is conserved between mouse and man.