Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.     

Purification and characterization of heparin-binding endothelial cell growth factors

Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.     

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.     

The contribution of endogenous and exogenous effects to radiation-induced damage in the bacterial spore

Radical scavengers such as polyethylene glycol 400 and 4000 and bovine albumin have been used to define the contribution of exogenous and endogenous effects to the gamma-radiation-induced damage in aqueous buffered suspensions of Bacillus pumilus spores. The results indicate that this damage in the bacterial spore is predominantly endogenous both in the presence of 1 atmosphere of oxygen, and in anoxia.     

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.