The role of intracellular glutathione in the progression of Chlamydia trachomatis infection

The productive internalization in the host cell of Chlamydia trachomatis elementary bodies and their infectivity depends on the degree of reduction of disulfide bonds in the outer envelope of the elementary body. We have hypothesized that the reducing agent may be intracellular glutathione (GSH). Three approaches were used to modulate the intracellular GSH concentration: (1) treatment of cells with buthionine sulfoximine, which causes irreversible inhibition of GSH biosynthesis; (2) hydrogen peroxide-induced oxidation of GSH by intracellular glutathione peroxidases; and (3) treatment of cells with N-acetyl-l-cysteine (NAC), a precursor of glutathione. In the first two cases, we observed a four- to sixfold inhibition of C. trachomatis infection, whereas in NAC-treated cells we detected an increase in the size of chlamydial inclusions. Using a proteomics approach, we showed that the inhibition of chlamydial infection does not combine with alterations in protein expression patterns after cell treatment. These results suggest that GSH plays a key role in the reduction of disulfide bonds in the C. trachomatis outer envelope at an initial stage of the infection.     

Gas-phase molecular structures of substituted 1,3-bisketenes: A challenge for theory and experiment

Molecular structures of dimethylbis(trimethylsilylketyl)silane (Me₂Si[C(SiMe₃)CO]₂), dimethylbis(trimethylgermylketyl)silane (Me₂Si[C(GeMe₃)CO]₂), and dimethylbis(trimethylstannylketyl)germane (Me₂Ge[C(SnMe₃)CO]₂) have been studied in the gas phase by electron diffraction accompanied by high level ab initio and DFT calculations. Extensive theoretical conformational analyses of the molecules in the vapour predicted a possibility of existence of two types of conformers with small energy differences. The first type had gauche-gauche arrangements of the ketenyl groups in the central C(CO)XC(CO) fragments directed away from each other. The second type had nearly syn-gauche arrangements of the ketenyl groups. In addition, the energy differences were found to depend on the level of computations used. The experimental analysis, in turn, was unable to distinguish between different conformers due to the large number of similar overlapping distances. The experimental data were fitted by an averaged single-conformer model, which nevertheless allowed reliable determination of bonds and bonded angles in the molecules. Main experimental (r_h1) structural parameters for Me₂Si[C(SiMe₃)CO]₂, Me₂Si[C(GeMe₃)CO]₂, and Me₂Ge[C(SnMe₃)CO]₂, i.e. Me₂X[C(YMe₃)CO]₂ (X,Y = Si, Ge, Sn), are (X-C)_mean 187.7(1) pm, 194.6(2) pm, 216.1(3) pm; (Y-C)_mean, 187.7(1) pm, 188.8(8) pm, 194.6(4) pm; (CC)_mean, 135.3(5) pm, 131.6(5) pm, 131.5(13) pm; (CO)_mean, 117.0(7) pm, 117.4(7) pm, 119.0(11) pm; (C-H)_mean, 110.6(7) pm, 110.0(4) pm, 109.1(13) pm; (X(Y)-CC)_mean, 114.4(2)°, 115.6(1)°, 115.6(2)°; (C-X(Y)-C_Me)_mean, 108.3(3)°, 108.4(3)°, 108.9(13)°; C(2)-C(1)-Y(4)-C(10), −19(6)°, 5(4)°, −9(10)°; C(7)-C(6)-Y(9)-C(38),−22(7)°, −32(3)°, −9(10)°; C(2)-C(1)-X(5)-C(6), 128(4)°, 142(1)°, 108(9)°; C(7)-C(6)-X(5)-C(1), 92(6)°, 115(2)°, 108(9)°, respectively.     

Protonation of lysine residues inverts cation/anion selectivity in a model channel

A dimeric alamethicin analog with lysine at position 18 in the sequence (alm-K18) was previously shown to form stable anion-selective channels in membranes at pH 7.0 [Starostin, A. V., R. Butan, V. Borisenko, D. A. James, H. Wenschuh, M. S. Sansom, and G. A. Woolley. 1999. Biochemistry. 38:6144-6150]. To probe the charge state of the conducting channel and how this might influence cation versus anion selectivity, we performed a series of single-channel selectivity measurements at different pH values. At pH 7.0 and below, only anion-selective channels were found with P(K(+))/P(Cl(-)) = 0. 25. From pH 8-10, a mixture of anion-selective, non-selective, and cation-selective channels was found. At pH > 11 only cation-selective channels were found with P(K(+))/P(Cl(-)) = 4. In contrast, native alamethicin-Q18 channels (with Gln in place of Lys at position 18) were cation-selective (P(K(+))/P(Cl(-)) = 4) at all pH values. Continuum electrostatics calculations were then carried out using an octameric model of the alm-K18 channel embedded in a low dielectric slab to simulate a membrane. Although the calculations can account for the apparent pK(a) of the channel, they fail to correctly predict the degree of selectivity. Although a switch from cation- to anion-selectivity as the channel becomes protonated is indicated, the degree of anion-selectivity is severely overestimated, suggesting that the continuum approach does not adequately represent some aspect of the electrostatics of permeation in these channels. Side-chain conformational changes upon protonation, conformational changes, and deprotonation caused by permeating cations and counterion binding by lysine residues upon protonation are considered as possible sources of the overestimation.     

Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells

Cyclo-oxygenase-2 (COX-2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX-2-catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX-2-associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX-2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX-2 in a cell-free system. Using spin-traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide and 4-((9-acridinecarbonyl) amino)-2,2,6,6- tetramethylpiperidine-1-oxyl (Ac-Tempo), we observed arachidonic acid (AA)-dependent production of carbon-centered radicals by heme-reconstituted recombinant COX-2. No oxygen radicals or thiyl radicals have been detected. COX-2 also catalyzed AA-dependent one-electron co-oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX-2 expression in cells, PCXII cells which express isopropyl-1-thio-beta-D-galactopyranoside inducible COX-2, and PC12 cells transfected with COX-2 using a protein delivery reagent, Chariot. In both models, COX-2-dependent AA-induced generation of carbon-centered radicals was documented using spin-traps and Ac-Tempo. No oxygen radical formation was detected in COX-2-transfected cells by either spin-traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA-induced COX-2-dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX-2 but was co-oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX-2 inhibitors, niflumic acid, nimesulide, or NS-398. Thus, COX-2 generated carbon-centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA-challenged PC12 cells overexpressing COX-2.     

Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine

The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.     

Understanding pH-dependent selectivity of alamethicin K18 channels by computer simulation

Alamethicin K18 is a covalently linked alamethicin dimer in which the glutamine residue at position 18 in each helix has been replaced by a lysine residue. As described in previous work, channels formed by this peptide show pH-dependent selectivity. The maximum anion selectivity of the putative octameric conducting state is obtained at pH 7 or lower. Inasmuch as no change in selectivity is seen between pH 7 and pH 3, and because protons are expected to be in equilibrium with the open state of the channel during a selectivity measurement, the channel is believed to be fully charged (i.e., all eight lysines protonated) at pH 7. In an effort to understand how such a highly charged channel structure is stable in membranes and why it is not more selective for anions, we have performed a number of computer simulations of the system. Molecular dynamics simulations of 10 ns each of the octameric bundle in a lipid bilayer environment are presented, with either zero, four, or eight lysines charged in the absence of salt, and with eight lysines charged in the presence of 0.5 M and 1 M KCl. When no salt is present and all lysines are charged, on average 1.9 Cl(-) ions are inside the channel and the channel significantly deforms. With 0.5 M KCl present, 2.9 Cl(-) ions are inside the channel. With 1 M KCl present, four Cl(-) ions are present and the channel maintains a regular structure. Poisson-Boltzmann calculations on models of the octameric channel also predict an average of 2-4 Cl(-) ions near the lysine residues as a function of ionic strength. These counterions lower the apparent charge of the channel, which may underlie the decrease in selectivity observed experimentally with increasing salt concentrations. We suggest that to increase the selectivity of Alm K18 channels, positive charges could be engineered in a narrower part of the channel.     

Simultaneous optical and electrical recording of single gramicidin channels

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.     

Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.     

Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation

The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels.     

Intensity of the humoral immune response to herpesviruses as an indicator the body immune deficiency

The state of the local immunity system of the oral cavity was studied by the level of saliva immunoglobulins in patients with different processes on their mucous membranes: herpetic infection, respiratory allergosis and malignant tumors of the mouth cavity and the laryngopharynx. The suppression of the production of sIgA, was accompanied by the enhanced production of antibodies to the most widespread herpesviruses (herpes simplex virus, cytomegalovirus and Epstein-Barr virus). The maximum levels of serum IgG to herpesviruses were determined in patients with malignant tumors. The role of herpesviruses in the pathogenesis of immunodeficient states is discussed.