Novel mono- and dicationic pyrimidinic surfactants are synthesized and their aggregation behavior is studied by methods of tensiometry and nuclear magnetic resonance (NMR) self-diffusion. To estimate their potentiality as gene delivery agents, the complexation with oligonucleotides (ONus) is explored by dynamic light scattering (DLS) and zeta-potential titration methods and ethidium bromide exclusion experiments. Bola-type pyrimidinic amphiphile (BPM) demonstrates rather a weak affinity to ONus. Although it induces mixed associations with ONus, only slight charge compensation changes occur at a large excess of bola, with no recharging reached. Similarly, the ethydium bromide exclusion study reveals a slow increase in the binding capacity toward an ONu with an increment in BPM concentration. The monocationic pyrimidinic surfactant (MPM) and its gemini analogue (GPM-1) are ranked as intermediates in both their aggregative activity and complexing properties toward ONus. They both form mixed associates with ONus well below the critical micelle concentrations (cmcs) of 2 and 15 mM respectively. However, GPM-1 has a much lower isoelectric point at the molar ratio surfactant/ONu r~1 compared to r~3 for MPM. This probably indicates a larger electrostatic contribution to the ONu complexation in the case of GPM-1. The most hydrophobic pyrimidinic surfactant (GPM-2), bearing three alkyl tails, demonstrates enhanced aggregative activity and binding capacity toward ONus as compared to former pyrimidinic surfactants. Due to effective aggregative (low cmc of 0.04 mM) plus binding properties (fraction of bound ONu β=0.76 at r=2.5), GPM-2 may be ranked as a promising agent for wider biological applications.

AIM The main purpose of this study was to estimate the SLC34A2 gene expression in normal ovary and different types of ovarian tumors. METHODS We have investigated SLC34A2 gene expression level in papillary serous, endometrioid, unspecified adenocarcinomas, benign tumors, and normal ovarian tissues using real-time PCR analysis. Differences in gene expression were calculated as fold changes in gene expression in ovarian carcinomas and benign tumors compared to normal ovary. RESULTS We have found that SLC34A2 gene was highly expressed in well-differentiated endometrioid and papillary serous ovarian carcinomas compared to low-differentiated endometrioid carcinomas, benign serous cystoadenomas and normal ovary. Analysis of SLC34A2 gene expression according to tumor differentiation level (poor- and well-differentiated) showed that SLC34A2 is up-regulated in well differentiated tumors. CONCLUSION Upregulation of SLC34A2 gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis.

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic,(10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that:(1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings;(2) the HPL products may have the regulatory role both in growth control and adaptation.

Lipoxygenases (LOXs) are key enzymes in the biosynthesis of oxylipins, the diverse class of bioregulators involved into developmental processes, signalling and defence. This work was undertaken to better understand how LOXs control production of hydroperoxides with different positional and stereochemistry. A number of glycerolipids were tested as substrates for maize 9-LOX (ZmLOX) and its A562G mutant form. Both the wild type (WT) ZmLOX and A562G mutant were shown to dioxygenate monolinolenoylglycerol (MLG) and 2-linoleoyl-sn-glycero-3-phosphorylcholine (lysoPC). Both the WT ZmLOX and A562G mutant form oxidized the MLG predominantly into (9S)-hydroperoxide. The A562G mutation did not affect the relative yield of 13-hydroperoxide, but increased the proportion of (13R)-enantiomer. LysoPC was a poor substrate for both wild type and A562G mutant form of ZmLOX. The oxidation of lysoPC exhibited the limited regio- and stereospecificity. Nevertheless, the WT ZmLOX produced some predominance of (13S)-hydroperoxide. In contrast, the A562G mutant produced some excess of (9S)-hydroperoxide of lysoPC. The bulky polar heads of glycerolipids like MLG and lysoPC cannot penetrate into the LOX active site. Thus, the obtained data indicate that both (9S)- and (13S)-hydroperoxides can be produced when substrate is arranged within LOX active site in the "methyl end first" orientation.

Expression of maize 9-lipoxygenase was performed and optimized in Escherichia coli Rosetta(DE3)pLysS. The purity of recombinant protein obtained during Q-Sepharose and Octyl-Sepharose chromatographies in an LP system at 4 degrees C was >95%. Maximum activity of the lipoxygenase reaction was observed at pH 7.5. Enzyme stability was studied at pH 4.5 to 9.5 and in the presence of different compounds: phenylmethanesulfonyl fluoride, beta-mercaptoethanol, ammonium sulfate, and glycerol. HPLC and GC-MS analysis showed that enzyme produced 99% 9S-hydroperoxide from linoleic acid. 13-Hydroperoxide (less than 1%) consisted of S- and R-enantiomers in ratio 2 : 3.

Abstract Erwinia carotovora ssp. atroseptica SCRI1043 is a plant pathogenic bacterium. Many species of enterobacteria including Erwinia sp. can induce the onset or the progression of opportunistic or persistent infections in humans. The existence of these bacteria within different ecological niches is related to their significant adaptive potential. The triggering of adaptive reactions, which are needed for bacterial persistence, is controlled in many cases by intercellular communication; hence, the ability to survive under unfavourable conditions is regulated in a cell-density-dependent manner. In this study, we showed that, during starvation of E. carotovora ssp. atroseptica SCRI1043, the initial stage of the response to stress was stabilization of the density of culturable cells in the range 10(6)-10(7) CFU mL(-1). The number of culturable cells increased (up to approximately 10(6) CFU mL(-1)) when cultures were inoculated at a low cell density (10(3)-10(5) CFU mL(-1)), and at the same time, acylhomoserine lactone (AHL)-dependent quorum sensing was activated. Our results showed that the regulation of cell density in starving populations of E. carotovora ssp. atroseptica SCRI1043 occurred and this regulation was carried out with the involvement of the cell-to-cell communication.

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (omega6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the "pocket theory of lipoxygenase catalysis"(i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.

The response to hydration of the internal protein dynamics was studied by the means of solid state NMR relaxation and magic angle spinning exchange techniques. Two proteins, lysozyme from bacteriophage T4 and human alphaB-crystallin were used as exemplars. The relaxation rates R(1) and R(1rho) of (13)C and (15)N nuclei were measured as a function of a hydration level of the proteins in the range 0-0.6 g of water/g of protein. Both proteins were totally (15)N-enriched with natural (13)C abundance. The relaxation rates were measured for different spectral bands (peaks) that enabled the characterization of the dynamics separately for the backbone, side chains, and CH(3) and NH(3)(+) groups. The data obtained allowed a comparative analysis of the hydration response of the protein dynamics in different frequency ranges and different sites in the protein for two different proteins and two magnetic nuclei. The most important result is a demonstration of a qualitatively different response to hydration of the internal dynamics in different frequency ranges. The amplitude of the fast (nanosecond time scale) motion gradually increases with increasing hydration, whereas that of the slow (microsecond time scale) motion increases only until the hydration level 0.2-0.3 g of water/g of protein and then shows almost no hydration dependence. The reason for such a difference is discussed in terms of the different physical natures of these two dynamic processes. Backbone and side chain nuclei show the same features of the response of dynamics with hydration despite the fact that the backbone motional amplitudes are much smaller than those of side chains. Although T4 lysozyme and alphaB-crystallin possess rather different structural and biochemical properties, both proteins show qualitatively very similar hydration responses. In addition to the internal motions, exchange NMR data enabled the identification of one more type of motion in the millisecond to second time scale that appears only at high hydration levels. This motion was attributed to the restricted librations of the protein as a whole.