The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Radiolysis of DNA in aqueous solution in the presence of a scavenger: A kinetic model based on a nonhomogeneous reaction of OH radicals with DNA molecules of spherical or cylindrical shape

Summary Two expressions are given for the survival dose of DNA exposed to high-energy radiation in aqueous solution in the presence of a scavenger. They are derived from a model where a diffusion controlled reaction of OH radicals occurs on the surface of the DNA macromolecules in competition with scavenging in the bulk of the solution. The DNA molecules are approximated either by spheres or by cylinders. The model based on molecules of spherical shape corresponds closely to that developed by van Rijn et al. [20]. Expressions obtained from the cylindrical model are used to account for the dependence on the scavenger concentration of some experimentally measured quantities, namely the survival dose and theG value for single-strand breaks upon Co -irradiation ofX 174 DNA and polyadenylic acid, respectively.In memoriam Prof. Dr. O.E. Polansky     

The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.     

Transmembrane signaling: tumor promoter distribution

Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.     

Nucleotide and deduced amino acid sequence of the hydrophobic surfactant protein SP-C from rat: expression in alveolar type II cells and homology with SP-C from other species

Pulmonary surfactant lowers surface tension in the lung. Its deficiency leads to the severe physiologic abnormalities seen in the respiratory distress syndrome. The hydrophobic surfactant proteins, SP-B and SP-C, appear to be especially important in the surface-spreading characteristics of pulmonary surfactant. We report the nucleotide sequence of cDNA clones for rat SP-C and compare the deduced amino acid sequence for SP-C from several species. A highly conserved domain exists within the confines of mature human SP-C. An Eisenberg plot of this region predicts a membrane-associated helix. We also demonstrate by Northern analysis the tissue-specific expression of SP-C. A comparison of signal strength between total lung RNA and RNA derived from isolated type II cells supports the idea that most SP-C messenger RNA in total lung can be accounted for by that present in alveolar type II cells.     

Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.