Regulation of unbalanced redox homeostasis induced by the expression of wild-type HIV-1 viral protein R (NL4-3Vpr) in fission yeast.

The wild-type viral protein R (Vpr) of human immunodeficiency virus type 1 exerts multiple effects on cellular activities during infection, including the induction of cell cycle G2 arrest and the death of human cells and cells of the fission yeast Schizosaccharomyces pombe. In this study, wild-type Vpr (NL4-3Vpr) integrated as a single copy gene in S. pombe chromosome was used to investigate the molecular impact of Vpr on cellular oxidative stress. NL4-3Vpr triggered an atypical response in early (14-h), and a wellregulated oxidative stress response in late (35-h) log-phase cultures. Specifically, NL4-3Vpr expression induced oxidative stress in the 14-h cultures leading, to decreased levels of superoxide anion (O(2)(·-)), hydroxyl radical (·OH) and glutathione (GSH), and significantly decreased activities of catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase. In the 35-h cultures, elevated levels of O(2)(·-) and peroxides were accompanied by increased activities of most antioxidant enzymes, suggesting that the Vpr-induced unbalanced redox state of the cells might contribute to the adverse effects in HIV-infected patients.

Antifungal activity of the primycin complex and its main components A1, A2 and C1 on a Candida albicans clinical isolate, and their effects on the dynamic plasma membrane changes.

The in vitro antifungal activities of the macrolide lactone antibiotic complex primycin (PC) and its main components, A1 (50%), A2 (7.3%) and C1 (13%), against the opportunistic pathogenic fungus Candida albicans 33erg(+)were determined by microdilution testing. The MIC(100) (the minimal concentration required for 100% growth inhibition) values found, A2 (2 µg ml(-1)), PC (32 µg ml(-1)), A1 (32 µg ml(-1)) and C1 (64 µg ml(-1)), suggested that the biological activity of PC is highly dependent on the proportions of its constituents. In vivo measurements of the biophysical properties of plasma membranes were carried out by electron paramagnetic resonance (EPR) spectroscopic methods, using the spin probe 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid. Conventional EPR measurements demonstrated altered phase transition temperatures (Tm) of the plasma membrane of strain 33erg(+) as a consequence of treatment with PC or its constituents: for cells treated with 128 µg ml(-1) PC, A1, A2 or C1 for 15 min, Tm was 17, 21, 14.5 and 15 °C, respectively; that is significantly higher than the Tm of untreated cells, 12 °C. The molecular motions of the near-surface hydrophobic region of the plasma membrane, estimated by saturation transfer EPR spectroscopy, reflected changes in the membrane phases after the treatment. Two physiological membrane phases were detected in control samples: liquid-ordered and liquid-disordered, characterized by molecular movements ∼10(-6)-10(-8) s and ≥10(-9) s. The cells treated with the investigated compounds showed the strong presence of a non-physiological gel phase additional to the above phases, characterized by movements ≤10(-5) s.

Direct in vivo interaction of the antibiotic primycin with the plasma membrane of Candida albicans: an EPR study.

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg(+) and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64 µgml(-1) for 33erg(+) and 128 µgml(-1) for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg(+) and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128 µgml(-1) primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg(+) and erg-2 were 60 ns and 100 ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8 µs and 1 µs. The results indicate the plasma membrane "rigidizing" effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.

The abc1-/coq8- respiratory-deficient mutant of Schizosaccharomyces pombe suffers from glutathione underproduction and hyperaccumulates Cd2+.

The abc1(-)/coq8(-) gene deletion respiratory-deficient mutant NBp17 of fission yeast Schizosaccharomyces pombe displayed a phenotypic fermentation pattern with enhanced production of glycerol and acetate, and also possessed oxidative stress-sensitive phenotypes to H(2)O(2), menadione, tBuOOH, Cd(2+), and chromate in comparison with its parental respiratory-competent strain HNT. As a consequence of internal stress-inducing mutation, adaptation processes to restore the redox homeostasis of mutant NBp17 cells were detected in minimal glucose medium. Mutant NBp17 produced significantly increased amounts of O(2)•- and H(2)O(2) as a result of the decreased internal glutathione concentration and the only slightly increased glutathione reductase activity. The Cr(VI) reduction capacity and hence the •OH production ability were decreased. The mutant cells demonstrated increased specific activities of superoxide dismutases and glutathione reductase (but not catalase) to detoxify at least partially the overproduction of reactive oxygen species. All these features may be explained by the decreased redox capacity of the mutant cells. Most notably, mutant NBp17 hyperaccumulated yellow CdS.

Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis.

BACKGROUND: Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC). RESULTS: SETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 +/- 0.7 degrees C, 57.9 +/- 0.7 degrees C, 63.7 +/- 1.0 degrees C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66-77 degrees C. CONCLUSION: According to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0-10.0 degrees C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.

Pro-oxidative versus antioxidative reactions between Trolox and Cr(VI): The role of H(2)O(2).

The effect of the Vitamin E model compound Trolox in reactions with Cr(VI) in the presence or absence of hydrogen peroxide was investigated. The aim of this study was to establish and discuss potential Trolox-mediated pro-oxidative reactions. The importance of the Trolox:Cr(VI) ratio in the Cr(VI) reduction process was determined from the EPR spectra and DNA cleavage reactions. In the absence of hydrogen peroxide, reduction of Cr(VI) occurred with concomitant oxidation of Trolox to the phenoxyl radical. In the presence of hydrogen peroxide, Cr(V), produced by the reduction of Cr(VI), reduced hydrogen peroxide to the hydroxyl radical. The latter was detected by spin-trapping the methyl radical following reaction with N-methyl sulfoxide. During Cr(VI) reduction with Trolox, DNA single- or double-strand breaks due to Trolox radical formation were not observed. Relaxed DNA appeared only when H(2)O(2) was added to Trolox/Cr(VI) mixtures most probably due to hydroxyl radical formation during the redox cycling of Cr(V/IV)-species. Fenton-like reactions do not play a significant role in the Trolox/Cr(VI) system in the absence of added H(2)O(2).

Effect of polycyclic aromatic hydrocarbons on erythrocyte membranes by DSC and EPR.

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on human erythrocyte membranes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model systems in order to study the effect the polycyclic aromatic hydrocarbons on lipid structure and dynamics. Eight different compounds among others naphthalene and pyrene were compared, which occur in significant concentrations in dust collected from the air in large cities. Experiments using spin label technique showed that the compounds induced mobility changes in the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes. The effects depended on the structure and concentration of the different compounds. Similarly to EPR observations, DSC measurements reported decrease of transition temperature in comparison to control DPPC vesicles. These results suggest that polycyclic aromatic hydrocarbons were able to modify the internal dynamics of erythrocyte membranes which might lead to damage of the biological functions.

Effects of hexavalent chromium on the plasma membranes of sensitive and tolerant mutants of Schizosaccharomyces pombe. An EPR study.

The interactions of chromium(VI) with the plasma membranes of chromium-sensitive (chr-51S) and chromium-tolerant (chr1-66T) mutants and their parental strain (6chr(+)) of a Schizosaccharomyces pombe strain were studied by electron paramagnetic resonance (EPR) spectroscopy. 5-doxylstearic acid (5-SASL) and 3-doxylbutyric acid (HO-185) spin probes were used to label the membranes. The order parameter S from the EPR spectra was calculated at different temperatures (0-25 degrees C) in order to characterize the internal dynamics of the membranes. In control experiments, both mutants exhibited differences in structural transitions in the both 5-SASL- and the HO-185-labeled membranes in comparison with their parental strain, suggesting differences in the membrane composition and/or rotational dynamics of these mutants. Addition of K(2)Cr(2)O(7) (225 microM) induced small decreases in the phase transition temperatures of the 5-SASL-labeled membranes of the parental and chromium-sensitive strains. More pronounced effects of the chromium compound on the HO-185-labeled membranes were detected as evidence that the membrane perturbations are mostly localized in the environment of the lipid-water interface.

Effect of nucleotides and their analogues on essential light chains in myosin head.

The domain movement in myosin head plays a decisive role in the energy transduction process of the muscle contraction. During hydrolysis of ATP, the specific formation of strong binding of myosin head for actin causes conformational changes. As a consequence, the light chain-binding motif generates the powerstroke. In our work maleimide spin labels were covalently attached to Cys-177 residue of ELC in subfragment-1 (S1). Our goal was to study the orientation dependence and the motion of S1, which were incorporated into glycerinated skeletal muscle fibres. The electron paramagnetic resonance spectroscopy (EPR) spectra of the probes depended strongly on the orientation of the fibre axis relative to the magnetic field, indicating that the essential light chain (ELC) and the neck were ordered. The probes were undergoing rapid motion within a cone. The half-width of the cone was estimated to be 65+/-5 degrees (SD, n=8). Addition of ADP affected little the hyperfine splitting and the angular spread of the probe distribution. In the presence of ADP and orthovanadate the intensity of the spectra decreased, which showed the dissociation of S1 and this was accompanied with the disappearance of the orientation dependence.

Effect of adenosine 5'-[beta,gamma-imido]triphosphate on myosin head domain movements.

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity. Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation-dependence of spin labels in the ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP-myosin, and the other half showed a different dynamic state with greater mobility.