Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Production of Cricotopus ornatus (Meigen) (Diptera: Chironomidae) in Waldsea Lake,Saskatchewan

Cricotopus ornatus was the predominant chironomid in meromictic, saline Waldsea Lake. Annual production of C. ornatus larvae in the mixolimnion was estimated to be 107 mg m^-2 (dry weight) in 1974, 66.5 mg m^-2 in 1975 and 69.5 mg m^-2 in 1976. These estimates are similar to those for chironomids in Canadian arctic lakes and deep-water areas of the Great Lakes. Annual P/B ratios were 5.4 in 1974, 6.8 in 1975 and 6.8 in 1976. These ratios are in the middle of the range reported for chironomids. The major factors limiting chironomid production in Waldsea Lake appear to be: (1) restriction of the habitable zone because of meromixis with accompanying loss of mobile first and second instars that are swept out of the mixolimnion (2) the relatively narrow zone of good C. ornatus habitat, i.e. areas of dense macrophyte or benthic algal growth and (3) predation by nine-spine stickleback and damselfly naiads.     

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.     

Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).     

A possible role of protein kinase C in signal-induced lysosomal enzyme release

In platelets, activation of protein kinase C and mobilization of Ca2+ were selectively induced by the addition of 1-oleoyl-2-acetyl-glycerol and a low concentration of A23187, respectively (Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704). Using this procedure evidence was obtained suggesting that the protein phosphorylation and Ca2+ mobilization were both essential and synergistically effective to cause release of lysosomal acid hydrolases such as N-acetylglucosaminidase. A similar observation was made for the lysosomal enzyme release from rat neutrophils.     

Control of protein synthesis in a wheat germ cell-free system

Thermostable low molecular weight translational inhibitor was found in wheat germ cell-free extract. The inhibitor was formed during preincubation of wheat S-23 fraction with components of the energy-supplying system (ATP, GTP, phosphoenolpyruvate) in the absence of exogenous mRNA. The inhibitor effectively and irreversibly blocks protein synthesis in both wheat germ and rabbit reticulocyte systems. Our results seem to suggest that the inhibitor can activate wheat endogenous mRNA, which under the standard conditions does not reveal template activity but, once activated, can effectively compete with exogenous mRNA.     

Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor

The ligand-binding activity of the galactose/N-acetylgalactosamine-specific receptor (Gal/GalNAc receptor) present on the surface of hepatocytes can be modulated under a number of conditions in the intact cell. The carboxylic acid ionophores monensin and nigericin inhibit endocytosis by the Gal/GalNAc receptor in a concentration-dependent manner. Monensin at a concentration of 100 microM reduces the number of binding sites for asialo-orosomucoid and a tri-branched glycopeptide (F2) 5-10-fold; however, the number of Gal/GalNAc receptor subunits detected at the cell surface by a competitive radioimmunoassay and by immunoprecipitation of surface labeled receptor is not significantly altered. Replacement of NaCl in the medium with either N-methylglucamine or sorbitol to isotonicity also inhibits binding and endocytosis. The monensin, nigericin, N-methylglucamine, and sorbitol treatments have in common the ability to alkalinize the cytosol of the hepatocyte. None of these agents has any effect on binding by the isolated Gal/GalNAc receptor nor is the intracellular pH shift of such a magnitude that it would alter binding by the isolated Gal/GalNAc receptor. This has led us to conclude that the ligand-binding properties of the Gal/GalNAc receptor at the cell surface can be modulated in a transmembrane fashion by events other than those involving pH or Ca2+ regulation at the ligand-binding site itself. Such transmembrane modulation of ligand binding by the Gal/GalNAc receptor may provide a rapid and efficient mechanism for mediating ligand release and immediate return of the receptor to the cell surface.     

Complex carbohydrates of cultured PC12 pheochromocytoma cells. Effects of nerve growth factor and comparison with neonatal and mature rat brain

The composition and biosynthesis of glycoproteins, proteoglycans, and gangliosides have been studied in a clonal line of rat pheochromocytoma (PC12) cells. Glycoproteins account for approximately 78% of the glucosamine-labeled complex carbohydrates found in the culture medium, together with 17% chondroitin sulfate and 5% heparan sulfate. 10% of the glycoproteins but less than 1% of the proteoglycans are released by trypsin treatment of the cells, whose complex carbohydrates are composed of 93% glycoproteins, 1.3% chondroitin sulfate, 3.4% heparan sulfate, and 2.6% of mono- and disialogangliosides. Sequential lectin affinity chromatography and alkali treatment of glycopeptides prepared from the medium, trypsin-releasable, membrane, and cell-soluble glycoproteins demonstrated that in all of the subfractions large tri- and tetraantennary complex oligosaccharides account for 82 to 97% of those present in PC12 cell glycoproteins. Biantennary oligosaccharides account for approximately 2-6% of those in medium and trypsinate, as compared to 10-13% in the membrane and cell soluble glycoproteins, and there were large differences (ranging from 7 to 60%) in the proportions of biantennary oligosaccharides which are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. High mannose oligosaccharides are present predominantly in the cell membrane and soluble glycoproteins, where they account for 4 to 5% of the total glycoprotein labeling. In response to nerve growth factor (NGF), the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. Significant alterations were also observed in the complex carbohydrates of NGF-treated cells, the most striking of which were an almost 3-fold increase in labeled gangliosides and a 75% increase in trypsin-releasable glycoproteins. Cellular heparan sulfate decreased by 70% in response to NGF and increased by an equivalent amount in the culture medium, whereas an NGF-induced increase in chondroitin sulfate labeling occurred specifically in the cell membranes.     

Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.