Tetraspanins as regulators of protein trafficking

Small transmembrane proteins of the tetraspanin superfamily are believed to function as the main structural blocks of specialized membrane microdomains (referred to as tetraspanin-enriched microdomains, TERM or TEM). Through a multitude of homotypic and heterotypic interactions, tetraspanins regulate lateral clustering and, consequently, signalling involving adhesion and growth factor receptors as well as costimulatory proteins. The presence of major histocompatibility complex (MHC) I and MHCII molecules in TERM led to suggestion of tetraspanins' involvement in antigen presentation. In addition, certain tetraspanins function as viral co-receptors and may be important for viral egress from infected cells. It has recently become apparent that in addition to their purely structural function as organizers of TERM, tetraspanins also regulate various aspects of trafficking and biosynthetic processing of associated receptors. Here, we review recent studies, which specifically focus on this issue.     

High-performance liquid chromatographic method with UV photodiode-array, fluorescence and mass spectrometric detection for simultaneous determination of galantamine and its phase I metabolites in biological samples

Galantamine, an alkaloid isolated from the bulbs and flowers of Caucasian snowdrop (Galanthus woronowii, Amaryllidaceae) and related species, is employed in human medicine for the treatment of various neuromuscular and neurodegenerative diseases. After the administration, the products of oxidative biotransformation (O-desmethyl-galantamine, N-desmethyl-galantamine, galantamine-N-oxide) and chiral conversion (epigalantamine) are formed in various concentrations from parent compound. For the identification and determination of galantamine and its phase I metabolites in blood plasma and tissues, a new bioanalytical method based on a reversed-phase high-performance liquid chromatography with UV photodiode-array, fluorescence and mass spectrometric detection was developed, validated and applied to pharmacokinetic and biotransformation studies. Sample preparation included a homogenization of the rat tissues (liver, brain, hypophysis) in a phosphate buffer 0.05 mol/L pH 7.4. Plasma samples and tissue homogenates were purified using a mixed-mode solid-phase extraction (Waters Oasis MCX cartridges). Galantamine, its above-mentioned metabolites and the internal standard codeine were separated on a Discovery HS F5 column (Supelco, 150 mmx4.6 mm I.D., 5 microm) at flow rate of 1 mL/min using a linear gradient elution. UV photodiode-array and mass spectrometric detection were employed for the identification of individual galantamine metabolites in various biomatrices, the fluorescence detection (lambdaexcit=280 nm/lambdaemiss=310 nm) was chosen for the quantification of galantamine and its metabolites. The developed method was applicable in liver tissue in the range from 0.50 to 63.47 nmol/g of galantamine, from 0.32 to 41.42 nmol/g of O-desmethyl-galantamine, from 0.54 to 69.40 nmol/g of N-desmethyl-galantamine and from 0.70 to 89.03 nmol/g of epigalantamine. Limit of detection was found to be 0.04 nmol/g for galantamine, 0.19 nmol/g for O-desmethyl-galantamine, and 0.07 nmol/g for N-desmethyl-galantamine and epigalantamine.     

Ribotyping and biotyping of<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolated from hospital environment

The Staphylococcus strains acquired from scrapings from hospital environments were identified to the species level based on their biochemical properties. From the monitored sample the Staphylococcus epidermidis strains were selected for more accurate typing and tested on their virulence factor and ribotyped. The biotyping of S. epidermidis did not show any considerable intraspecific variation of these isolates and there were no atypical reactions, with the exception of three strains (out of 33). In contrast, the results of ribotyping showed greater heterogeneity of strains and unequivocally demonstrated the relation between the ribotype and the place of sample drawing. In addition to this fact, the found ribotypes repeat in the same environment in the long-term which suggests the occurrence and persistence of the same strains of conditionally pathogenic bacteria in hospital environment. We showed that ribotyping is a suitable method for precise and reliable detection of some coagulase-negative staphylococci.     

Role of extracellular reactive sulfur metabolites on microbial Se(0) dissolution

The dissolution of elemental selenium [Se(0)] during chemical weathering is an important step in the global selenium cycle. While microorganisms have been shown to play a key role in selenium dissolution in soils, the mechanisms of microbial selenium solubilization are poorly understood. In this study, we isolated a Bacillus species, designated as strain JG17, that exhibited the ability to dissolve Se(0) under oxic conditions and neutral pH. Growth of JG17 in a defined medium resulted in the production and accumulation of extracellular compounds that mediated Se(0) dissolution. Analysis of the spent medium revealed the presence of extracellular sulfite, sulfide, and thiosulfate. Abiotic Se(0) dissolution experiments with concentrations of sulfite, sulfide, and thiosulfate relevant to our system showed similar extents of selenium solubilization as the spent medium. Together, these results indicate that the solubilization of Se(0) by JG17 occurs via the release of extracellular inorganic sulfur compounds followed by chemical dissolution of Se(0) by the reactive sulfur metabolites. Our findings suggest that the production of reactive sulfur metabolites by soil microorganisms and the formation of soluble selenosulfur complexes can promote selenium mobilization during chemical weathering.     

The whither of bacteriophytochrome‐based near‐infrared fluorescent proteins: Insights from two‐photon absorption spectroscopy

We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors.      

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

Amiodarone inhibits multiple drug resistance in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.     

Changes in inflammatory activity,heart rate variability,and biochemical indices in young athletes during the annual training cycle

Inflammatory activity, heart rate variability (HRV), and biochemical and functional indices were assessed in young ski racers during the preparation, competition, and recovery periods of an annual training cycle. During the preparation period, autohemodilution (decreased red blood cell count (RBC) and hematocrit (Ht) levels) and a decrease in the systemic inflammatory activity (C-reactive protein) occurred, without significant differences in the HRV or serum protein and lipid profile. During the competition period, the systemic inflammatory activity increased by 50% (p = 0.047), eliminating differences from the control group, and the HRV indices (SDNN, HF, TP, and IT) decreased (p ≤ 0.013), indicating an increase in the sympathetic effects on the HRV. During the recovery period, hematological indices (Ht and RBC), inflammatory activity, and fibrinogen levels decreased, and the parasympathetic effects on the heart increased. These findings allowed us to conclude that mental and physical activation led to a moderate increase in the systemic inflammatory activity and a shift in the sympathovagal balance towards increased sympathetic activity, providing a nonspecific contribution to the physiological regulation of biochemical (lipoproteins and immunoglobulins) and hematological indices in the athletes. However, similar changes in many biochemical and hematological indices in both groups during the year indicated the important role of a common, probably seasonal, factor in the regulation.     

Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273–1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663–668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH₂ interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ^? being reduced back to SkQH₂ by heme b_H of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C₉ or C₁₃ of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (С₁₂TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since С₁₂TPP is specifically accumulated in mitochondria and increases the H⁺ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by С₁₂TPP.