Triton X-100 partitioning into sphingomyelin bilayers at subsolubilizing detergent concentrations: effect of lipid phase and a comparison with dipalmitoylphosphatidylcholine

We examined the partitioning of the nonionic detergent Triton X-100 at subsolubilizing concentrations into bilayers of either egg sphingomyelin (SM), palmitoyl SM, or dipalmitoylphosphatidylcholine. SM is known to require less detergent than phosphatidylcholine to achieve the same extent of solubilization, and for all three phospholipids solubilization is temperature dependent. In addition, the three lipids exhibit a gel-fluid phase transition in the 38-41 degrees C temperature range. Experiments have been performed at Triton X-100 concentrations well below the critical micellar concentration, so that only detergent monomers have to be considered. Lipid/detergent mol ratios were never <10:1, thus ensuring that the solubilization stage was never reached. Isothermal titration calorimetry, DSC, and infrared, fluorescence, and (31)P-NMR spectroscopies were applied in the 5-55 degrees C temperature range. The results show that, irrespective of the chemical nature of the lipid, DeltaG degrees of partitioning remained in the range of -27 kJ/mol lipid in the gel phase and of -30 kJ/mol lipid in the fluid phase. This small difference cannot account for the observed phase-dependent differences in solubilization. Such virtually constant DeltaG degrees occurred as a result of the compensation of enthalpic and entropic components, which varied with both temperature and lipid composition. Consequently, the observed different susceptibilities to solubilization cannot be attributed to differential binding but to further events in the solubilization process, e.g., bilayer saturability by detergent or propensity to form lipid-detergent mixed micelles. The data here shed light on the relatively unexplored early stages of membrane solubilization and open new ways to understand the phenomenon of membrane resistance toward detergent solubilization.     

Proteomic analysis of CD44(+) and CD44(−) gastric cancer cells

CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(?) gastric cancer cells to understand characteristics of CD44(+) and CD44(?) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(?) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(?) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(?) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(?) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(?) cells and one protein (prohibitin) showed increased expression in CD44(?) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(?) cells in gastric cancer.     

Comparisons of population density and genetic diversity in artificial and wild populations of an arborescent coral,Acropora yongei: implications for the efficacy of “artificial spawning hotspots”

We are developing techniques to restore coral populations by enhancing larval supply using “artificial spawning hotspots” that aggregate conspecific adult corals. However, no data were available regarding how natural larval supply from wild coral populations is influenced by fertilization rate and how this is in turn affected by local population density and genetic diversity. Therefore, we assessed population density and genetic diversity of a wild, arborescent coral, Acropora yongei, and compared these parameters with those of an artificially established A. yongei population in the field. The population density of wild arborescent corals was only 0.27% of that in the artificial population, even in a high‐coverage area. Genetic diversity was also low in the wild population compared with the artificial population, and approximately 10% of all wild colonies were clones. Based on these results, the larval supply in the artificial population was estimated to be at least 1,400 times higher than that in wild A. yongei populations for the same area of adult population.     

Tetracycline-resistant bacteria as indicators of antimicrobial resistance in protected waters—The example of the Drwęca River Nature Reserve (Poland)

The objective of this study was to determine the suitability of Tet^R tetracycline-resistant bacteria as potential indicators of drug resistance, a parameter of the microbiological quality of river waters in natural reserves which are threatened by man-made pollution. The microbiological assays covered a 15-km long section of the upper reach of the Drw?ca River (Poland), a part of the European Ecological “Natura 2000” Network of nature protected areas. The quality of the investigated ecosystem was affected by surface runoffs from the river's agricultural catchment as well as outflows from three fish farms. The counts of Tet^R bacteria, incubated at 14 °C and 28 °C on TSA medium with sheep blood and tetracycline, were determined in river water samples. The highest counts of both bacterial groups were determined in samples collected from sites behind fish farms. A statistical analysis of the abundance of Tet^R14 °C and Tet^R28 °C bacteria revealed significant differences in the size of Tet^R28 °C populations at the studied sampling sites (p = 0.0011), which is why hemolytic bacteria of this group (HemTet^R28 °C) were selected for further investigations. The predominant strains in the group of 86 HemTet^R28 °C isolates obtained by 16S rRNA gene sequencing were Pseudomonas fluorescens (42 isolates) and Aeromonas hydrophila (28 isolates). Analyses of the identified HemTet^R28 °C strains demonstrated MIC ≥256 μg/ml in more than 50% isolates. The MAR index of HemTet^R28 °C was in the range of 0.67 at the control site to 1 at sites behind fish farms. The results suggest that tetracycline-resistant bacteria, in particular HemTet^R28 °C, are a reliable indicator of antimicrobial resistance and the microbial quality of surface waters polluted due to human activity. The above can be attributed to several factors: (I) the highest percentage share of HemTet^R28 °C among HPC28 °C was noted at sites behind fish farms, (II) tetracycline-resistant bacteria quickly respond to environmental changes, as demonstrated by the high level of resistance to tetracycline and a very high MAR index, and (III) genera/species that are easy to culture under laboratory conditions dominate in the qualitative and quantitative composition of the studied bacteria.     

Release of dialkylglycerol from purple membrane phospholipids by phospholipase D

When the major polar lipid of purple membrane, a dialkyl analogue of phosphatidyl glycerophosphate, is treated with phospholipase D under the usual assay conditions for this enzyme, the reaction yields dialkylglycerol and glycerol bisphosphate, i.e. the kind of products that would be expected from a phospholipase C reaction. The effect is seen both in native purple membranes and with the pure phospholipid in the form of liposomes. The specific activity and kinetic parameters Km and Vmax of phospholipase D for the purple membrane phospholipid are similar to those for egg phosphatidylcholine. The presence of phospholipase C impurities in the phospholipase D preparations has been ruled out as an explanation for the above observations. A hypothesis is suggested, taking into account the peculiar headgroup structure of the bacterial lipid, to explain the seemingly anomalous enzyme behavior.     

Kinetics of purple membrane dark-adaptation in the presence of Triton X-100

The kinetics of purple membrane dark adaptation were studied at pH 5 and 7, in the presence and absence of the nonionic detergent Triton X-100. The effect of both sublytic and lytic surfactant concentrations has been considered. Our results show that: (a) dark adaptation is faster at pH 5 than at pH 7, (b) dark adaptation is slower, and of smaller amplitude, in the presence than in the absence of Triton X-100. The data may be interpreted in terms of a simple first-order kinetic model, according to which light-dark adaptation would depend basically on the equilibrium between the 13-cis- and the all-trans-isomers. The experiments also suggest that at pH 5, but not at pH 7, solubilizing surfactant concentrations produce a considerable increase in the velocity of the dark adaptation reaction, perhaps through changes in the microenvironment of a protonable group.     

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.     

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5′-dithio-bis-(2-nitrobenzoic acid) and ZnCl₂, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H₂O₂. SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.