The Release and Detection of Endotoxin from Liposomes

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulateLimulusamebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxinin vitro.This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C₁₂E₁₀) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C₁₂E₁₀varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.     

Colonization of a novel depauperate habitat leads to trophic niche shifts in three desert lizard species

In a novel, depauperate ecosystem, colonizing species may experience changes in their trophic niche as a result of a new resource base and fewer competitors and predators. To examine trophic niche shifts of recent colonists, we focused on three ecologically and phylogenetically divergent lizard species that inhabit both the geologically distinctive depauperate habitat of White Sands and the surrounding Chihuahuan ‘dark soil’ desert in New Mexico. In White Sands the three species comprise the entire lizard community, whereas in the dark soils habitat, they constitute less than half of the lizard community abundance. As a result, we hypothesized that the three focal species would collectively represent a greater variety of trophic positions in the White Sands habitat than in the dark soils habitat. We hypothesized that the extent of shifts in each species’ trophic position would parallel diet and ecomorphology differences between habitats. To test these hypotheses, we combined analysis of lizard stomach contents with carbon and nitrogen stable isotopes in the context of previously published ecomorphology measurements. Stable isotope data indicated that as predicted, species were more different from one another in White Sands than in dark soils, suggesting community‐wide ecological release. Overall, all species were lower on the White Sands food chain; however, only one species decreased trophic level significantly, one increased trophic level variance, and one did not change significantly. Furthermore, stomach content data paralleled both stable isotope and ecomorphological data, showing different degrees of dietary overlap between habitats, depending on the species. That species’ differences in trophic ecology also correspond with ecomorphological differences suggests that these factors are either causally linked or collectively responding to similar ecological pressures, such as competition. By examining diet, trophic position, and ecomorphology of three colonist species, we demonstrate both species‐specific and community‐wide trophic differences in adjacent, but distinct habitats.     

A procedure for detecting phosphonolipids on thin-layer chromatograms

A simple modification of a phospholipid-specific spray for thin-layer chromatograms allows it to be used as a specific detection reagent for phosphonolipids and as a general lipid detection reagent.     

Biosynthesis of N-(purin-6-ylcarbamoyl)-l-threonine riboside. Incorporation of l-threonine in vivo into modified nucleoside of transfer ribonucleic acid

l-[U-(14)C]Threonine is incorporated into N-(purin-6-ylcarbamoyl)-l-threonine riboside of rat liver and Escherichia coli tRNA. A pathway is suggested for the biosynthesis of this nucleoside.     

Characterization of Purified Staphylococcal Lipase

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the alpha, beta, and alpha' positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme.     

Thermal Resistance of Staphylococcus aureus in Milk, Whey, and Phosphate Buffer

The thermal resistance of four strains of coagulase-positive Staphylococcus aureus was determined in phosphate buffer, whole milk, skim milk, and Cheddar cheese whey. The logarithmic order of death prevailed until about 99.99 to 99.999% of the organisms were destroyed, after which there was a decline in the rate of destruction. The organisms were more resistant in skim milk and Cheddar cheese whey than in phosphate buffer and whole milk. Thermal resistance varied among strains of S. aureus but was consistent with individual strains. As the age of cultures of strain B-120 increased from 12 to 228 hr, the D(55) values increased from 0.95 to 3.0. The thermal resistance of cultures obtained from survivors to partial thermal destruction was similar to that of the parent cultures.     

Integrating ecology into macroevolutionary research

On 9 March, over 150 biologists gathered in London for the Centre for Ecology and Evolution spring symposium, 'Integrating Ecology into Macroevolutionary Research'. The event brought together researchers from London-based institutions alongside others from across the UK, Europe and North America for a day of talks. The meeting highlighted methodological advances and recent analyses of exemplar datasets focusing on the exploration of the role of ecological processes in shaping macroevolutionary patterns.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Altered accumulation of a membrane protein unique to a membrane-deoxyribonucleic acid complex in a dna initiation mutant of Bacillus subtilis.

Membrane-deoxyribonucleic acid complexes (M-bands) have been isolated from Bacillus subtilis by their affinity for crystals of Mg2+-Sarkosyl. The membrane proteins of these complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the membrane protein composition of M-band and unfractionated membrane revealed three protein components of 125,000 (mac-1), 57,000 (mac-2), and 42,000 (mac-3) daltons unique to M-band membrane. Growth of a temperature-sensitive dna initiation mutant at the restrictive temperature resulted in an accumulation in the membrane of mac-2. This accumulation did not begin, however, until cell growth had nearly ceased, some 3 to 4 h after the cessation of deoxyribonucleic acid synthesis. Upon return of the mutant to the permissive temperature, mac-2 did not begin to return to normal levels until after the first round of deoxyribonucleic acid synthesis. A protein of 30,000 daltons, common to both M-band and whole membrane, was found to disappear from the membrane when the mutant was grown at the restrictive temperature. This disappearance is the result of increased degradation or removal from the membrane followed by a decreased rate of synthesis or insertion.