Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

Influence of Meloidogyne incognita on Resistant and Susceptible Sweet Potato Cultivars

Effects of several population densities ofMeloidogyne incognita on the sweet potato cultivars Centennial (susceptible) and Jasper (moderately resistant) were studied. Field plots were infested with initial levels (Pi) of 0, 10, 100, 1,000, 5,000, and 10,000 eggs and juveniles/500 cm³ soil in 1980 and 0, 100, 1,000, 2,000, 3,000, 4,000, and 5,000 in 1981. M. incognita population development trends were similar on both cultivars; however, at high Pi, more eggs and juveniles were recovered from Centennial than from Jasper. The highest Pi did not result in the highest mid-season (Pm) counts. Pi was negatively correlated with the number of marketable roots and root weight but positively correlated with total cracked roots, percentage of cracked roots, and cracking severity. Jasper tolerated higher Pi with greater yields and better root quality than Centennial. Cracking of fleshy roots occurred with both cultivars at low Pi.     

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast

Summary We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.     

Isozymes of phosphorylase kinase in rabbit skeletal muscle. Functional implications of differences in phosphorylase kinase and phosphorylase activities in individual muscle fibers

Immunological and microanalytical methods were used to investigate the two isozymes of phosphorylase kinase, enzyme w and enzyme r, in psoas major and tibialis anterior muscles. Peptide mapping experiments indicated that the alpha subunit of enzyme w and alpha' subunit of enzyme r were structurally very similar. Both subunits were completely immunoprecipitated from muscle extracts with an antibody specific for the beta subunit of the kinase, indicating that alpha and alpha' subunits are completely assembled with beta subunits in adult muscle fibers. The relative amounts of enzymes w and r in single fibers were determined from amounts of alpha and alpha' subunits, which were detected by immunoblotting. Phosphorylase kinase and phosphorylase activities were measured in the same fibers, as well as in individual fibers from diaphragm and soleus muscles. Slow oxidative fibers were found to contain low levels of enzyme r, but almost no enzyme w. Considerably more enzyme r was present in fast oxidative-glycolytic fibers. Fast glycolytic fibers contained the most enzyme w, and the highest levels of enzyme r were found in a subgroup of such fibers. Interestingly, more than half of the fast glycolytic fibers analyzed contained both isozymes. In these fibers phosphorylase was positively correlated with enzyme w, but negatively correlated with enzyme r. Total kinase activity ranged 30-fold from the highest in one of the psoas fibers to the lowest in one of the soleus fibers and was closely correlated with the phosphorylase levels. In psoas and soleus fibers, calculated absolute maximal rates for phosphorylase b to a conversion varied almost 2,500-fold.     

Combinations of palytoxin or 12-O-tetradecanoylphorbol-13-acetate and recombinant human insulin growth factor-I or insulin synergistically stimulate prostaglandin production in cultured rat liver cells and squirrel monkey aorta smooth muscle cells

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) or the non-TPA-type tumor promoter, palytoxin, recombinant human insulin growth factor-I (IGF-I) and insulin synergistically stimulate prostaglandin production in rat liver cells (the C-9 cell line). Combinations fo palytoxin or TPA with recombinant human IGF-I or insulin also synergistically stimulate deesterification of c ellular lipids in C-9 cells prelabelled with [³H]arachidonic acid. With both types of stimulations, prostaglandin production or deesterification, the synergistic response of the IGF-I and insulin is greater with palytoxin than with TPA. Production of prostaglandin E₂ and F_2α by squirrel monkey smooth muscle cells incubated in the presence of TPA and insulin also is greater than the sum of the effects taken independently.     

Effects of bacterial endotoxin on protecting copper-deficient rats from hyperoxia

The administration of very low doses of bacterial endotoxin protects rats during exposure to hyperoxia and is associated with the induction of lung antioxidant enzyme activities. Copper-deficient rats have increased susceptibility to O2 toxicity, which may be related to their decreased lung superoxide dismutase activity (SOD) or decreased plasma ceruloplasmin concentrations. To determine whether endotoxin can protect against hyperoxia in this susceptible model, we exposed copper-deficient and control rats to a fractional inspiratory concentration of O2 greater than 0.95 for 96 h after pretreatment with 500 micrograms/kg of bacterial endotoxin or phosphate-buffered saline (PBS). Mortality in the copper-deficient and control rats given PBS and exposed to O2 for 96 h was 100%. Copper-deficient rats died significantly earlier during the exposure than controls. No mortality occurred in either group treated with endotoxin and hyperoxia despite the decreased activity of copper-dependent enzymes in the copper-deficient rats. Copper-deficient rats treated with endotoxin and exposed to hyperoxia did increase lung Cu-Zn-SOD activity, but activity remained below levels found in air-exposed controls. Mn-SOD activity was found to be induced above air-exposed controls in the copper-deficient rats treated with endotoxin and exposed to hyperoxia. Hyperoxic exposure resulted in a marked increase in plasma ceruloplasmin concentrations in the control rats, but no increases in ceruloplasmin occurred in the copper-deficient animals. Endotoxin protects copper-deficient rats from hyperoxia despite their decreased lung Cu-Zn-SOD activity, and decreased plasma ceruloplasmin.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

Calmodulin in interferon preparations: effect of interferon on calmodulin bioactivity

Heat-stable calmodulin immunoreactivity and bioactivity were detected in crude preparations of various types of human, murine and chicken interferons (IFNs). Calmodulin containing HuIFN-alpha was retained on a trifluorophenothiazine-Sepharose column. The two activities were separated by serial elutions with 50 microM Ca2+ (HuIFN-alpha) followed by 2 mM EGTA (calmodulin). While maintaining its full antiviral activity, calmodulin free HuIFN-alpha inhibited enhancement of Ca2+-ATPase activity in vitro by authentic purified eukaryote calmodulin. These results indicate that IFNs are calmodulin-binding proteins and that the secretion of both IFNs and calmodulin occurs from IFN-induced cells.