Abraham H. Parola

Phosphatidylethanolamine and phosphatidylglycerol are segregated into different domains in bacterial membrane. A study with pyrene‐labelled phospholipids

To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene‐labelled phospholipids, 2‐pyrene‐decanoyl‐phosphatidylethanolamine (PY‐PE) and 2‐pyrene‐decanoyl‐phosphatidylglycerol (PY‐PG) were inserted into Escherichia coli or Bacillus subtilis membrane. The excimerization rate coefficient, calculated from the excimer‐to‐monomer ratio dependencies on the probe concentration, was two times higher for PY‐PE than for PY‐PG at 37°C. This was ascribed to different local concentrations rather than to differences in mobility. The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E. coli and B. subtilis, in contrast to model membranes. In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order. Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes. It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo–lipid interaction and degree of order; the proteo‐lipid domain being enriched by PE.

Transcription‐ and translation‐dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation

Cell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion (‘transertion’) of nascent membrane and exported proteins. Disruption of domain structure should lead to changes in membrane dynamics. Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain. The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later. The drug‐induced decrease in membrane viscosity is energy dependent: it did not happen in KCN‐treated cells. All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion. Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand. Changes in membrane dynamics were also displayed during a nutritional shift‐down transition that causes imbalance in macromolecular syntheses. The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is ‘frozen’.

Low-frequency electromagnetic fields induce a stress effect upon higher plants, as evident by the universal stress signal, alanine

15N NMR analysis reveals alanine production in Duckweed plants exposed to low intensity sinusoidally varying magnetic fields (SVMF) at 60 and 100Hz, and fed by 15N-labeled ammonium chloride. Alanine does not accumulate in the absence of SVMF. Addition of vitamin C, a radical scavenger, reduced alanine production by 82%, indicating the roll of free radicals in the process. Alanine accumulation in plants and animals in response to exposure to a variety of stress conditions, including SVMF, is a general phenomenon. It is proposed that alanine is a universal first stress signal expressed by cells.

Coexistence of Domains with Distinct Order and Polarity in Fluid Bacterial Membranes

In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan‐labeled Escherichia coli and temperature dependencies of both laurdans GP and fluorescence anisotropy of 1,3‐diphenyl‐1,3,5‐hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid‐crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37°C reflects membrane heterogeneity. Time‐resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdans emission. In the proteo‐lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple‐Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid‐crystalline bacterial membrane: a lipid‐enriched and a proteo‐lipid domain. In bacteria with chloramphenicol (Cam)–inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature‐dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam‐induced domain dissipation.

Effect of sinusoidally varying magnetic fields on cell proliferation and adenosine deaminase specific activity

The effect of sinusoidally varying magnetic fields (SVMF) on chick embryo fibroblasts (CEF) was examined by two independent methods: 1) measurement of cell proliferation at 0.06-0.7 mT (100, 60 and 50 Hz) using a colorimetric assay (MTT); 2) monitoring of specific activity of adenosine deaminase (ADA) at 0.3 and 0.7 mT, 60 Hz. Both increased cell proliferation and reduced ADA specific activity are associated with cell transformation. The MTT test showed an increase in cell proliferation of up to 64% after a 24 h exposure to SVMF at 100 Hz, 0.7 mT. Cell proliferation at constant frequency (100 Hz) depended on SVMF intensity. Cell proliferation at constant intensity (0.7 mT) increased with increasing field frequency. At 0.7 mT, 60 Hz cell proliferation increased by 31%, 28%, and 26% when measured by hemocytometry, 3H-thymidine incorporation, and the MTT assay, respectively. ADA specific activity in CEF decreased by circa 48% on exposure to SVMF at 60 Hz, 0.3 mT for 24 h; only a statistically insignificant trend was seen at 0.7 mT, 60 Hz. Our findings showed that CEF cell proliferation and ADA specific activity were modified by SVMF. Both methods, independently, qualitatively detect a magnetic field effect.

Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential

To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.

Homeoviscous adaptation, growth rate, and morphogenesis in bacteria.

Fluorescence polarization, P, of 1,6-diphenyl-1,3,5-hexatriene was studied in Escherichia coli B/r. Modification of nutritional conditions was not compensated by homeoviscous adaptation, demonstrated to exist for temperature variations. Cell diameter, which is known also to vary with nutrition but not with temperature, was found to be positively correlated with 1/P, and may therefore be regulated by membrane lipid order and fluidity.

Novel fluorescence-photochrome labeling method in the study of biomembrane dynamics

A novel photochrome-fluorescence method (PFLM) based on monitoring fluorescence parameters and kinetics of photochrome photoisomerization of para-substituted stilbenes (PSS) has been proposed. It was shown that PSS exhibits fluorescence characteristics which are similar to ones of typical membrane fluorescence probes such as diphenylhexatriene (DPH). A study of kinetics of PSS trans-cis and cis-trans photoisomerization makes it possible to estimate, under certain conditions, the rotational correlation time of the stilbene fragments in the excited state of PSS for the fixed angle 180 degrees. In viscous media this process is a rate-determining stage. Taken together, the both techniques, fluorescence and photochrome, make it possible to establish a detailed mechanism and measure quantitative parameters of stilbene probe (PSS) mobility in a membrane. The PFLM was applied to the study of E. coli membrane dynamics.

Membrane dynamic alterations associated with activation of human platelets by thrombin.

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.

Membrane dynamic alterations associated with viral transformation and reversion: Decay of fluorescence emission and anisotropy studies of 3T3 cells

Abstract Fluorescence lifetimes, anisotropies and rotational correlation time values of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of normal, transformed, and revertant 3T3 cells were determined by nanosecond (nsec), photon counting spectrofluorimetry. No change in lifetime values with transformation or reversion is observed. Fluorescence anisotropy decay curves show at least two components; an initial relatively fast decay and a non-zero “plateau” level component. The observed changes in the average anisotropy values, which qualitatively follow steady-state fluorescence polarization values, is due primarily to changes in the non-zero “plateau” level component. The anisotropy decay curves suggest that the rotational motion of the probe is restricted to a limited angular range. The present results are compared with model membrane systems.