Jaroslava Svobodová

Review of surfactin chemical properties and the potential biomedical applications

Surfactin, a highly powerful biosurfactant produced by various strains of the genus Bacillus, exhibits antibacterial, antiviral, antitumor and hemolytic action. This anionic cyclic lipopeptide is constituted by a heptapeptide interlinked with a β-hydroxy fatty acid. Due to its amhipathic nature surfactin incorporates into the phospholipid bilayer and induces permeabilization and perturbation of target cells. The rising antibiotic resistance as well as a number of remarkable surfactin activities shows that it deserves special interest and is considered as a candidate compound for combating several health related issues. In this review, the current state of knowledge of surfactin properties, biomedical potential and limitations for its application is presented.

Time-resolved polarized fluorescence studies of the temperature adaptation in Bacillus subtilis using DPH and TMA-DPH fluorescent probes

The validity of the concept of homeoviscous adaptation was tested for bacteria Bacillus subtilis. The Bacillus subtilis grown at 20 degrees C (referred to as Bs20) exhibit a considerable increase of branched anteiso-C15, the major fatty acid component of membrane lipids, relative to membranes grown at 40 degrees C (Bs40). The time-resolved fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) showed that these changes in the lipid composition are accompanied by changes in a mean lipid order. In particular, the DPH order parameters and measured in Bs20 membranes at 18 degrees C and in Bs40 membranes at 45 degrees C, respectively, tend to be equal. This effect was less pronounced for TMA-DPH. Our observations suggest that a physical parallel to the changes of lipid composition is the maintenance of an optimal lipid order in the hydrophobic core of the cytoplasmic membranes. It can be interpreted as a tendency of Bacillus subtilis to keep the lateral pressure in its membranes at an optimal value, independent of the temperature of cultivation.

Fluorescence decay and depolarization in membranes

In optically discontinuous and/or anisotropic environments such as lipid membranes, the radiative decay rate is dependent on the orientation of the emission dipole. A theory of fluorescence decay and depolarization in membranes has been developed that takes the orientational dependence into account. The theory predicts nonexponential total emission intensity decay and emission anisotropy decay different than the one in optically isotropic systems. 1,6‐diphenyl‐1,3,5‐hexatriene fluorescence in L‐α‐dipalmitoyl‐phosphatidylcholine large unilamellar vesicles has been analyzed in terms of this theory. It has been shown that the orientational dependence of the radiative rate is sufficient to account for the complexity of the intensity decay in membranes. The analysis also allowed the determination of the refractive index, orientational order, and rotational dynamics in the membrane.

DnaK and GroEL chaperones are recruited to the Bacillus subtilis membrane after short-term ethanol stress

Aims:  To find out membrane tolerance strategy to ethanol in Bacillus subtilis that possesses a powerful system of protection against environmental stresses.

Cold shock in Bacillus subtilis: different effects of benzyl alcohol and ethanol on the membrane organisation and cell adaptation.

A temperature shift-down of Bacillus subtilis from 40 to 20 degrees C induces an 80 min growth lag. Benzyl alcohol reduced this period to 51 min, whereas ethanol prolonged it up to 102 min. The effect of the two alcohols on the membrane state was investigated by measuring the steady-state fluorescence anisotropy and analysing the lifetime distribution of diphenylhexatriene (DPH) and its polar derivative, TMA-DPH. As followed from the fluorescence anisotropy, the two alcohols exerted similar (fluidizing) effects on the cytoplasmic membranes of B. subtilis. However, benzyl alcohol significantly shortened the main DPH lifetime component and widened its distribution, while ethanol had no effect. The benzyl alcohol activity was interpreted in terms of an increased membrane hydration due to disordering of the membrane structure. Such an effect imitates the cold shock induced synthesis of unsaturated fatty acids in B. subtilis. The fatty acid analysis revealed that ethanol hindered this adaptive synthesis of fatty acids. At the same time, its effect on the membrane state (membrane order) was very low and could not substitute the physiological response as was the case with benzyl alcohol. It can thus be concluded that the adaptation of the membrane physical state contributes significantly to the cold shock response of B. subtilis.

Membrane fluidity inBacillus subtilis. Physical change and biological adaptation

The thermotropic behaviour of membrane phospholipids was estimated in intact cells ofBacillus subtilis. Membrane fluidity (microviscosity) of intact cells depended markedly on the ambient temperaturo — increase in cultivation temperature led to an increase in membrane fluidity. Estimated as anisotropy of 1,6-diphenyl-1,3,5-hexatriene fluorescence, a 30 % difference was observed when cells cultivated at 20 and 40 °C were compared. This lack of rigorous homeostatic control of bulk-phase lipid fluidity prompted the reevaluation of the physiological significance of the “homeoviscous adaptation” inB. subtilis.

Membrane fluidity inBacillus subtilis. Validity of homeoviscous adaptation

The validity of the principle of homeoviscous adaptation forBacillus subtilis was tested by comparing fluorescence aniaotropy (1,6-diphenyl-1,3,5-hexatriene) and electron-spin resonance (16-doxylstearate) measurements carried out in isolated plasma membranes and in phospholipid fractions. The physical measurements were supplemented by fatty-acid analysis. The results support our previous findings on intact cells. The thermoadaptive mechanism ofB. subtilis manifested as an increase in relative proportion of branchedanteiso-C15 andanteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.

Pulse wave analysis during supine rest may identify subjects with recurrent vasovagal syncope

In the present study, we studied whether analysis of the FAP (finger arterial pressure) waveform during supine rest discriminates subjects with recurrent VVS (vasovagal syncope) from healthy controls. Signal-averaged FAP waveforms (Finapres) were obtained in 32 head-up tilt-test-positive subjects with recurrent VVS (35 +/- 13 years) and in 32 sex- and age-matched healthy controls. The DT (time delay) between the systolic and diastolic peaks of the FAP waveform was measured and large artery SI (stiffness index) was calculated as a ratio of body height and DT. VVS patients had significantly shorter DT compared with controls (303 +/- 31 compared with 329 +/- 18 ms; P < 0.001) and higher SI (5.79 +/- 0.70 compared with 5.20 +/- 0.36 m/s; P < 0.001). The differences were independent of heart rate and blood pressure. SI > 5.45 m/s identified subjects with syncope with a sensitivity of 72% and a specificity of 84%. Age-corrected DT (cDT = DT + age-350) identified subjects with syncope with a sensitivity of 75% and a specificity of 84%. Combined use of cDT <0 ms and SI > 5.45 m/s increased sensitivity and specificity to 81% and 96% respectively. The discriminative power of FAP descriptors improved further when younger subjects were excluded. In subjects aged >30 years (median age), the combination of cDT and SI identified subjects with syncope with a sensitivity of 93% and a specificity of 100%. These results suggest that FAP descriptors during supine rest might be useful in the diagnosis of VVS in middle-aged subjects.

Cytoplasmic membrane fluidity measurements on intact living cells ofBacillus subtilis by fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene

A method for determination of membrane fluidity (microviscosity) inBacillus subtilis cytoplasmic membrane underin vivo conditions is described. The membranes were labelled with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene during the exponential phase of growth. Fluorescence anisotropy values were determined as soon as 4 min after termination of the cultivation. Fluorescence anisotropy measurements were carried out in an intact cell suspension having absorbanceA as high as 0.2-0.3 (corresponding to a cell concentration of 100-300/nL).

Regulation of glycerol 3-phosphate oxidation in mitochondria by changes in membrane microviscosity

The inhibition of glycerol 3‐phosphate oxidation by oleic acid correlates with changes in membrane microviscosity monitored by the steady‐state fluorescence anisotropy of DPH. The dynamic measurements indicate that the changes of both the limiting anisotropy and rotational relaxation time occur in a concentration range where the enzyme activity is strongly inhibited.