Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

Network-Free Inference of Knockout Effects in Yeast

Perturbation experiments, in which a certain gene is knocked out and the expression levels of other genes are observed, constitute a fundamental step in uncovering the intricate wiring diagrams in the living cell and elucidating the causal roles of genes in signaling and regulation. Here we present a novel framework for analyzing large cohorts of gene knockout experiments and their genome-wide effects on expression levels. We devise clustering-like algorithms that identify groups of genes that behave similarly with respect to the knockout data, and utilize them to predict knockout effects and to annotate physical interactions between proteins as inhibiting or activating. Differing from previous approaches, our prediction approach does not depend on physical network information; the latter is used only for the annotation task. Consequently, it is both more efficient and of wider applicability than previous methods. We evaluate our approach using a large scale collection of gene knockout experiments in yeast, comparing it to the state-of-the-art SPINE algorithm. In cross validation tests, our algorithm exhibits superior prediction accuracy, while at the same time increasing the coverage by over 25-fold. Significant coverage gains are obtained also in the annotation of the physical network.     

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Requirement for extracellular calcium or magnesium in mitogen-induced activation of human peripheral blood lymphocytes

The importance of calcium in lymphocyte activation is well recognized, but the levels of extracellular ionized free calcium (Ca++) necessary for lymphocyte proliferation via various pathways have not been investigated in detail. We studied the ability of a lectin mitogen (PHA) and a calcium ionophore (ionomycin) to induce interleukin 2 receptors, interleukin 2 (IL2) production, and proliferation over various concentrations of extracellular Ca++. Reducing the Ca++ levels from the normal 200 microM to 10 microM in PHA-stimulated cultures partially inhibited IL2 receptor expression, IL2 production, and subsequent proliferation. At 1 microM Ca++, both IL2 activity and proliferation were eliminated, but partial IL2 receptor expression was still observed. Ionomycin did not induce any of these events in cultures where the extracellular Ca++ concentration was below 100 microM. Restoring calcium in the medium resulted in normal levels of IL2 receptor expression, IL2 activity, and proliferation when PBL were stimulated with either mitogen. Exogenous magnesium partially restored these events in PHA-stimulated cultures, but had no effect when ionomycin was used as the mitogen. These data indicate that stimulation by ionomycin is much more dependent upon the levels of extracellular Ca++ than is PHA. Extracellular calcium also appears to be necessary subsequent to IL2 receptor acquisition, since the latter was seen without IL2 activity or proliferation at very low extracellular Ca++, and IL2 failed to restore the proliferative response under these conditions. The data also suggest that PHA, but not ionomycin, can activate lymphocytes via a magnesium-dependent pathway, or that PHA has a lower specificity for divalent cation cofactors.     

Population and pharmacogenetic studies on serum cholinesterase (E): estimates of theE 1 andE 2 allele frequencies for the population of Central Italy,and of the correlation between E activity and sensitivity to succinylcholine (SCC)

392 random patients treated with SCC prior to surgery were assayed for pseudocholinesterase activity and electrophoretic pattern. The estimate of the percent frequencies of theE ₁ ^a allele were 1.16±0.38 and of the C₅ (+) phenotype 9.7±1.5, both typical of Caucasian populations. By combining the presentE ₁ ^a gene frequency estimate with that from another sample of the same population (Cermele et al., 1987) a better estimate with smaller confidence limits was obtained, that is: 1,14±0,27. One subject interpreted asE ₁ ^a E ₁ ^a on phenotypic grounds was also found (expected 0,05) in this random sample. A correlation coefficient of −0.521 was found between E activity and myorelaxation time in the whole sample. High correlation values (r=−0.55 and r=−0.46) were found forE ₁ ^u E ₁ ^u ; C₅ (−) andE ₁ ^u E ₁ ^u ; C₅ (+) individuals, respectively, showing a strong dependence of the latter variable on the former one even within apparently homogeneous classes. The use of the product of these two variables as a classification criterion allowed the identification of a subject with very long myorelaxation time but normal activity.     

Circadian variations in melatonin-binding sites in discrete areas of the male rat brain

The binding of 125I-melatonin to synaptosomes prepared from whole brains of male rats of the CD strain and from the brain, hypothalamus and striatum of male rats of the Sabra-Wistar strain was assessed throughout a 24 h period. The animals were maintained under a daily schedule of 14 h light (05:00-19:00 h) and 10 h darkness. In whole brain preparations the density of binding sites at 18:00 h was higher by about 70% than at 02:00 h with no variations in apparent affinity of the binding sites throughout the daily period. Specific binding of 125I-melatonin was found in both hypothalamus and striatum of the male rat with a distinct diurnal variation in binding site density in the hypothalamus only. The density of 125I-melatonin-binding sites in the hypothalamus was maximal between 10:00 and 18:00 h and dropped sharply after the lights went off. The apparent 125I-melatonin-binding affinities in these regions were constant and very similar to those in whole brain preparations. The daily variations in densities of 125I-melatonin-binding sites in discrete brain areas may represent a diurnal rhythmicity in the responsiveness of the neuroendocrine axis to melatonin.     

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.     

Mass action kinetics of virus-cell aggregation and fusion.

A simple approximate solution for the mass action kinetics of small particles (viruses or vesicles) binding to large particles (cells) and their subsequent fusion has been derived. The solution is evaluated in terms of the measurable fluorescence changes expected when the virus or vesicles are labeled with fluorescent probes, which are diluted into the cellular membrane by fusion. Comparison with numerical integrations shows that the approximate solution is extremely accurate. Analytic simplifications for a variety of special cases of this general problem are also shown.     

Precipitation of calcium palmitate from bile salt-containing dispersions

Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.