Cell suspensions for kinetic studies of inhibitor action

Because of uniformity and small distances for transport, cell suspensions offer a system for rapid measurements of initial reactions of phytotoxic compounds. We had previously shown that a growth regulator, dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-L-gulonate) inhibits amino acid incorporation into proteins. Using Solanum nigrum suspension cultures, it was found that dikegulac rapidly inhibits amino acid uptake into cells, before inhibiting incorporation, with time points starting at a few minutes, and kinetics that can be extrapolated back to time zero. With more rapid kinetics this compound induces leakage of a preloaded dye. The rate of leakage was less with stationary cells in suspension, reiterating that they are more resistant to the effects of this compound. It was thus concluded that at the concentrations used, the first effect of dikegulac (or one very close to the first effect) is on the cell membrane.Abbreviation FDA fluorescein diacetate     

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging,Cell Injury Measurements,and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

A hierarchy of lipid constructs for the sperm plasma membrane

We have presented a series of lipid constructs as models of the sperm plasma membrane. We also isolated the plasma membrane from rabbit sperm cells and characterized the lipid composition. The behavior of these various membrane systems was evaluated using a vesicle leakage assay, in which surfactant (nonoxynol-9, N-9; or benzalkonium chloride, BZK) exposure induced membrane permeabilization. These studies shed light on the relative importance and significance of particular components present in the lipid constructs. In particular, a highly unsaturated phospholipid component characterized by an ether-linkage to position 1 of the glycerol backbone (as opposed to the more conventional ester linkage) as well as the presence of sulfogalactosyl ceramide were found to have an effect on the surfactant-induced leakage response. The presence of cholesterol had the greatest influence on membrane behavior. The construct series also demonstrated the ability of the surfactants studied to discriminate between different membrane systems. We found that N-9 displayed little sensitivity to membrane composition while BZK showed specific behavior with the various membrane systems.     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

Tau Oligomers Impair Artificial Membrane Integrity and Cellular Viability

The microtubule-associated protein Tau is mainly expressed in neurons, where it binds and stabilizes microtubules. In Alzheimer disease and other tauopathies, Tau protein has a reduced affinity toward microtubules. As a consequence, Tau protein detaches from microtubules and eventually aggregates into β-sheet-containing filaments. The fibrillization of monomeric Tau to filaments is a multistep process that involves the formation of various aggregates, including spherical and protofibrillar oligomers. Previous concepts, primarily developed for Aβ and α-synuclein, propose these oligomeric intermediates as the primary cytotoxic species mediating their deleterious effects through membrane permeabilization. In the present study, we thus analyzed whether this concept can also be applied to Tau protein. To this end, viability and membrane integrity were assessed on SH-SY5Y neuroblastoma cells and artificial phospholipid vesicles, treated with Tau monomers, Tau aggregation intermediates, or Tau fibrils. Our findings suggest that oligomeric Tau aggregation intermediates are the most toxic compounds of Tau fibrillogenesis, which effectively decrease cell viability and increase phospholipid vesicle leakage. Our data integrate Tau protein into the class of amyloidogenic proteins and enforce the hypothesis of a common toxicity-mediating mechanism for amyloidogenic proteins.     

Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

EVect of Chitosan on Growth and Toxin Production by Alternaria alternata f. sp. lycopersici

The antifungal activity of chitosan, a biopolymer of beta-1-4 glucosamine, against Alternaria alternata f. sp. lycopersici , causal agent of black mold of tomato, was investigated. Chitosan was incorporated into potato-dextrose broth at concentrations of 100-6400 mug ml - 1, and the growth and toxin production by the fungus were assessed after 15 days of incubation. At the higher concentrations, chitosan significantly aVected both fungal growth and toxin production. However, at lower concentrations toxin production was aVected more than growth. The fungus sporulated excessively in the presence of chitosan, but the spores were less viable. Chitosan also induced aggregation, abnormal shape, excessive branching and hyphal contortion of fungal cells, and leakage of proteins. The virulence of the toxin in culture filtrates of the fungus grown on diVerent concentrations of chitosan was assessed by administering toxin on tomato disks. The phospholipid content, electrolyte leakage and activities of xylanase and pectin methylesterase were measured in the tomato tissue administered with culture filtrates containing fungal toxin. Decreased trends in the tendency to cause electrolyte leakage, phospholipid degradation and activation of xylanase and pectin methylesterase in the tomato tissue were observed with increasing concentrations of chitosan. The results showed that toxin produced in the presence of chitosan was less eVective in causing degradation of tomato tissue compared with the control. Thus, chitosan is a potential antifungal agent which can interfere with the pathogenic factors of the fungus.     

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.     

Chromium-induced production of reactive oxygen species,DNA single-strand breaks,nitric oxide production,and lactate dehydrogenase leakage in J774A.1 cell cultures

The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed. We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability. Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 μM for Cr (VI) and Cr (III), respectively. The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III). A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production. Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 μM. Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations. The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species. © 1996 John Wiley & Sons, Inc.