Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable of inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.     

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [³H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [³H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [³H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca²⁺ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.     

Liposome-mediated delivery of antibody to a Drosophila cell line

Large, unilamellar vesicles composed of equimolar amounts of acidic phosopholipids and phosphatidylethanolamine were able to deliver fluorescent dye [5(6)-carboxyfluorescein] or a monoclonal antibody directed against intermediate-filament proteins to a Drosophila cell line (Kc cells). Millimolar Ca2+ or protamine sulfate in microgram quantities triggered rapid, synchronous delivery of either solute. Delivery required a specific lipid composition: liposomes composed of 1:1 mole ratios of phosphatidylethanolamine:phosphatidylserine were able to deliver their contents, but not if phosphatidylcholine was substituted for phosphatidylethanolamine. Light microscopic observation of Kc cells incubated with free dye or antibody alone showed very little uptake, a result indicating that encapsulation within liposomes is a prerequisite for substantial delivery. Moreover, the stability of adhering vesicles in the absence of calcium or protamine sulfate, the lipid specificity, and the rapid onset of intracellular fluorescence after triggering suggest that vesicle-cell fusion is the predominant mode of solute uptake. Fusion of liposomes with the cell membrane was confirmed by freeze-fracture electron microscopy, which showed liposome vesicles first adhering to cell surfaces, then undergoing fusion when calcium or protamine sulfate was added.     

New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.     

Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions.

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.     

Base composition of deoxyribonucleic acid isolated from mycobacteria

Guanine plus cytosine values of deoxyribonucleic acid derived from 30 cultures representing 14 mycobacterial species or varieties are presented. These data provide impressive reasons for maintaining the separation between the genera Corynebacterium and Mycobacterium; no conclusions can be arrived at from these data with respect to the Nocardia-Mycobacterium relationship. A bimodal clustering, in terms of guanine plus cytosine composition, is apparent within the genus Mycobacterium. In general, all members of any single phenetic species appear to fit into one or another of these clusters. The phenetic separation of species is, in some cases, confirmed by separation in terms of guanine plus cytosine values. The bimodal separation of guanine plus cytosine values within the genus Mycobacterium does not correspond to a division of the species into slow and rapid growers; it thus provides no justification for splitting Mycobacterium into two genera, composed of slow and rapid growers. This is not to say that such a split would not be useful, only that these data do not contribute to such a decision. Any further attempts to correlate phenetic classification with properties of mycobacterial deoxyribonucleic acid will require more specific techniques, such as molecular hybridization.     

Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate, and its alpha and gamma substituents

We have examined the growth-inhibitory potency of several pteridines encapsulated in negatively charged liposomes, including methotrexate, methotrexate-gamma-methylamide, methotrexate-gamma-dimethylamide, methotrexate-alpha-aspartate, and a lipophilic methotrexate-phosphatidylethanolamine conjugate. The potency of encapsulated methotrexate is greater than the potency of the free drug for CV1-P cells, but not for other cell lines. The potency of methotrexate-gamma-methylamide and methotrexate-gamma-dimethylamide is only minimally improved by encapsulation. The potency of methotrexate-alpha-aspartate is increased by encapsulation. In addition, the lipophilic methotrexate derivative has demonstrable potency when incorporated in liposomes. We have also examined the potency of several pteridines under conditions where the cells are exposed to the drug for periods shorter than the entire growth assay. Reduction of the exposure time decreases the potency of both encapsulated and free drugs. However, the difference in potency between the encapsulated and free drug is increased, because the potency of the encapsulated drug is affected less. Consequently, encapsulated methotrexate-gamma-aspartate is 300-fold more potent than free drug, if CV1-P cells are exposed to drug for 4 h. Moreover, encapsulated methotrexate is more potent than free methotrexate for growth inhibition of L929 fibroblasts, if the term of exposure is less than 8 h. Potency is least affected by reduction of exposure length for the lipophilic methotrexate derivative.     

Phospholipid characteristics and neutral lipid fatty acid composition related to temperature and nutritional conditions in ecologically important amphipod species from the northern Baltic Sea

Lipid composition of the benthic, stenothermal amphipod Monoporeia affinis collected from constantly cold deep-water (> 80 m) regions of the northern Baltic Sea was analysed in detail to study phospholipid characteristics, its relation to thermal adaptation as well as potential effects of food quality and seasonal variability. Similar measurements were performed on the littoral, eurythermic amphipod Gammarus spp. Fatty acid (FA) composition of storage lipids of Pontoporeia femorata, a sibling species of M. affinis, was also studied. Interannual and interspecies variability in selected FA was observed both in triacylglycerols (TAG) and phospholipids (PL). Differences in monounsaturated vs. polyunsaturated (MUFA/PUFA) FA combinations of phosphatidyl ethanolamine (PE) diacyl and alkylacyl subgroups were also observed. Seasonal variability in M. affinis was considerably lesser compared to Gammarus spp. A literature synopsis shows that in diacyl PE the share of the FA combination MUFA/PUFA increases with lowering body temperature (40-55% in cold-adapted organisms vs. 5-15% in warm adapted ones), signifying that this characteristic is probably in key position concerning regulation of membrane fluidity in temperature adaptation. According to this feature M. affinis belongs to the group of cold-adapted stenothermic species (Group 1) while eurythermic Gammarus spp. settles in the Group 2 (cold-acclimatized) or 3 (warm-acclimatized/tropical) depending on the status of temperature adaptation. Omnivory and/or periodical food item switching is an important strategy in benthic organisms in high-latitude areas characterised by recurring long poor-nutritional periods. Since many benthic species utilise a time-averaged, integrated food source from phytal and animal matter from various sources the FA composition of TAG of the amphipod species measured here do not exclusively point towards any specific feeding mode or food source. In general, using selected FA as food source markers to assess the diet of field collected more-or-less omnivoric species cannot be considered as an optimal approach. The current study gives more insight to the biochemistry of biological membranes of aquatic crustaceans that is essential in estimation of the capacity of the thermal adaptation/acclimatization of organisms as well as the potential effects of food quality on storage lipids.     

Antigenic and deoxyribonucleic acid base composition of aMycoplasma and associated diphtheroids

Three diphtheroids isolated from cultures ofMycoplasma arthritidis (Campo strain) were shown to cross-react serologically with theMycoplasma. Stable L-forms prepared from these bacteria were shown to have still greater antigenic similarity to theMycoplasma. But the three diphtheroids isolated on three separate occasions were shown not to be identical antigenically. The buoyant densities of the DNA's of the L-forms were similar to those of the diphtheroids from which they were derived. The caesium chloride gradient method indicated a significant difference in nucleotide base composition between the purified deoxyribonucleic acid of the diphtheroids and of theMycoplasma.Junior Research Fellow supported by USPH training grant 5 ROI AI 00232.     

Liposome-mediated transfer of YAC DNA to tobacco cells

This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective.     

Analysis and sorting of living cells according to deoxyribonucleic acid content.

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.