Kinetics of cholesterol extraction from lipid membranes by methyl-beta-cyclodextrin--a surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-beta-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-beta-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.     

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.     

Receptors subtypes involved in adenosine-mediated modulation of norepinephrine release from cardiac nerve terminals

The objective of this study was to determine which adenosine receptor subtypes were involved in the modulation of norepinephrine release from cardiac nerve terminals. In addition, the persistence of adenosine-mediated effects was evaluated. Rat hearts attached to the stellate ganglion were isolated and perfused. The ganglion was electrically stimulated twice (S1 and S2), allowing 10 min between the stimulations. To determine adenosine receptor subtypes, selective and nonselective adenosine agonists and antagonists were infused following S1 and until the end of S2. To evaluate the persistence of adenosine-mediated effect on norepinephrine release, the stellate ganglion was stimulated a third (S3) and fourth (S4) time. Coronary effluents were collected to determine norepinephrine content. Adenosine and a selective A1 receptor agonist, CCPA, inhibited norepinephrine release by 49% and 54%, respectively. This effect was reversed by simultaneous infusion of nonspecific (8-SPT) and specific (DPCPX) A1 receptor antagonists. Selective A2A (CGS 21680) and A3 (AB-MECA) receptor agonists had no discernible effect on norepinephrine release. Similarly, adenosine A2A receptor antagonists CSC and DMPX did not alter the dose-response relation between norepinephrine release and adenosine. Finally, the inhibitory effects of adenosine on norepinephrine release did not persist 10 min subsequent to the removal of adenosine. Adenosine inhibited norepinephrine release primarily via the adenosine A1 receptor. This effect of adenosine was of short duration. Adenosine A2A and A3 receptors were either absent or functionally insignificant in the regulation of norepinephrine release in the rat heart.     

Novel thrombin inhibitors incorporating weakly basic heterobicyclic P1-arginine mimetics: optimization via modification of P1 and P3 moieties

Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P(1)-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors 17-29 and enantiomerically pure thrombin inhibitors 30-33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-ylamine.     

Characterization of quercetin binding site on DNA gyrase

Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a K(D) value of 15 microM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin-gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.     

Effective conductivity of a suspension of permeabilized cells: a theoretical analysis

During the electroporation cell membrane undergoes structural changes, which increase the membrane conductivity and consequently lead to a change in effective conductivity of a cell suspension. To correlate microscopic membrane changes to macroscopic changes in conductivity of a suspension, we analyzed the effective conductivity theoretically, using two different approaches: numerically, using the finite elements method; and analytically, by using the equivalence principle. We derived the equation, which connects membrane conductivity with effective conductivity of the cell suspension. The changes in effective conductivity were analyzed for different parameters: cell volume fraction, membrane and medium conductivity, critical transmembrane potential, and cell orientation. In our analysis we used a tensor form of the effective conductivity, thus taking into account the anisotropic nature of the cell electropermeabilization and rotation of the cells. To determine the effect of cell rotation, as questioned by some authors, the difference between conductivity of a cell suspension with normally distributed orientations and parallel orientation was also calculated, and determined to be <10%. The presented theory provides a theoretical basis for the analysis of measurements of the effective conductivity during electroporation.     

Antibody response to GroEL varies in patients with acute Mycoplasma pneumoniae infection

Although heat-shock proteins represent major antigens in a wide spectrum of bacterial infections, their immunogenicity is not known for Mycoplasma pneumoniae. M. pneumoniae is a major human respiratory pathogen and it has been suggested that its groEL gene might be dispensable in vitro. Using the specific monoclonal antibody 2C2/C3 we found an abundant synthesis of about 58 kDa GroEL in M. pneumoniae reference strains and in 15 clinical isolates examined at low and higher passages. In patients with acute respiratory disease caused by M. pneumoniae immunoblot analyses showed relatively low prevalence of systemic antibodies against its GroEL protein. Whereas all patients had strong antibody response to the P1 adhesin, only 5 of 29 patients (17.2%) had antibodies to GroEL. Among them, patient RI raised an early and very strong antibody response to GroEL. During the convalescent phase, levels of his serum IgG (mainly IgG2) to GroEL increased and were higher than levels of IgG to P1.     

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross-talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)-dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon-optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl-cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP-mediated, PKA-dependent phosphorylation. Further evidence for cross-talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar-apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA-dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.     

The response of giant phospholipid vesicles to pore-forming peptide melittin

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 microg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT-vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT-membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.