DNA damage and repair in Helicobacter pylori-infected gastric mucosa cells

Helicobacter pylori is a common human pathogen and its infection is believed to contribute to gastric cancer. Impaired DNA repair may fuel up cancer transformation by the accumulation of mutation and increased susceptibility to exogenous carcinogens. To evaluate the role of infection of H. pylori in DNA damage and repair we determined: (1) the level of endogenous basal, oxidative and alkylative DNA damage, and (2) the efficacy of removal of DNA damage induced by hydrogen peroxide and the antibiotic amoxicillin in the H. pylori-infected and non-infected GMCs. DNA damage and the efficacy of DNA repair were evaluated by the alkaline single cell gel electrophoresis (comet assay). Specific damage to the DNA bases were assayed with the DNA repair enzymes formamidopyrimidine-DNA glycosylase (Fpg) recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The level of basal and oxidative DNA in the infected GMCs was higher than non-infected cells. H. pylori-infected GMCs displayed enhanced susceptibility to hydrogen peroxide than control cells. There was no difference between the efficacy of DNA repair in the infected and non-infected cells after treatment with hydrogen peroxide and amoxicillin. Our results indicate that H. pylori infection may be correlated with oxidative DNA damage in GMCs. Therefore, these features can be considered as a risk marker for gastric cancer associated with H. pylori infection and the comet assay may be applied to evaluate this marker.     

Genotoxic activity of a technical toxaphene mixture and its photodegradation products in SOS genotoxicity tests

Toxaphene (CAS No. 800-35-2) is a complex mixture of several hundred components that was used worldwide primarily as an agricultural pesticide with insecticide effects in the second half of the 20th century. In vitro investigations of the genotoxicity and mutagenicity of toxaphene were generally described in the literature, but they provided somewhat equivocal results. We re-evaluated the genotoxicity of technical toxaphene in two prokaryotic systems. The SOS Chromotest showed high sensitivity to toxaphene: three concentrations (40, 20 and 10 mg/l) were clearly positive and the dose-response effect was evident. In the umuC assay, a dose-dependent increase in genotoxic activity was observed at toxaphene concentrations from 2.5 to 40.0 mg/l, but these results were found to be not significant. The genotoxicity of toxaphene and its photodegradation products after UV-irradiation (3-6-9 h) at concentrations ranging from 7.5 to 60.0 mg/l was also examined in this study. An irradiated solution of technical toxaphene after 3 h showed no significant evidence of bacterial growth inhibition. However, exposure of Salmonella to 6 h UV-irradiated toxaphene showed a toxic effect compared with the negative control. After 9 h irradiation, a decrease of bacterial growth was observed. Activity of beta-galactosidase in the presence of a toxaphene solution was significantly increased after 6 and 9 h irradiation, reaching values that were 2.4- and 3.1-fold higher, respectively, than the control, which exceeded the criteria of significant genotoxicity. These results show that while technical toxaphene is a weak, direct-acting mutagen in some bacterial tests, a dose-dependent toxicity and genotoxicity of its photoproducts could be conclusively demonstrated by the umuC test.     

Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp+ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 (sfp+) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.     

Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-gamma by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.     

Stereochemistry of internucleotide bond formation by the H-phosphonate method. 1. Synthesis and 31P NMR analysis of 16 diribonulceoside (3'-5')-H-phosphonates and the corresponding phosphorothioates

Sixteen diribonucleoside (3'-5')-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3'-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. 31P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arisingfrom Rp and Sp diastereomers were reversed.     

Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.     

Post-hatching dynamics of plasma biochemistry in free-living European starlings (Sturnus vulgaris)

This is the first study of plasma biochemical parameters in free-living altricial birds during an entire developmental period in a nest, represented by European starling (Sturnus vulgaris). Dynamics of postnatal changes from hatching until close to fledging (days 1 to 15) were registered. Parameters of protein metabolism represented by total proteins, albumin and globulin concentrations increased continuously during the observed developmental period. There were two peaks in uric acid concentration on days 5 and 11. To the contrary, the creatinine content did not change throughout the observed period and increased only on day 15. Creatine kinase activity gradually increased until day 11 and then fell before fledging. Parameters of lipid metabolism (concentration of total lipids, triacylglycerols and nonesterified fatty acids) in plasma increased gradually reaching a plateau between days 8 and 11 and then declined on day 15. The cholesterol concentration pattern was similar to maximum value on day 11, then consecutively decreased. Concentration of glucose increased until day 8 and remained unchanged until fledging. Whereas calcium reached the highest concentration during days 8 and 11, phosphorus peaked earlier on day 5. The activity of alkaline phosphatase was similar to the pattern found in calcium concentration. Presented data showed an increase in both protein and lipid metabolism during the phase of rapid growth. A remarkable decrease in parameters of lipid metabolism before fledging may reflect increased physical activity and changes in nutrition.     

Bryophyte and vascular plant responses to base-richness and water level gradients in Western Carpathian<Emphasis Type="Italic">Sphagnum</Emphasis>-rich mires

We investigated the importance of water chemistry and water regime for vascular plant and bryophyte species distribution in Western Carpathian mires dominated bySphagnum. Seventy-seven small circle plots distributed across a wide geographical area, a wide range of mineral richness and all possible microtopographical features were sampled in terms of species composition, physical-chemical water properties and water regime during one growing season. Both water chemistry and water regime were found to be important factors for vegetation composition. Bryophytes reflected only one clear gradient, connected to base-richness (pH, conductivity) and maximal water-level, whereas three different environmental gradients determined the occurrence of vascular plants: water-level amplitude, base-richness and an indistinct gradient presumably connected to peat layer thickness. When the entire data set was subjected to DCA ordination, the first resulting axis was governed by the bryophyte subset, whereas the second one was governed by the vascular plant subset. The species density of vascular plants was positively correlated with pH and conductivity. On the contrary, bryophyte species density showed no relationship to environmental factors. We further compared the pH values measured in groundwater and in water squeezed from bryophytes from the same plot; these plots were distributed along the base-richness gradient. Only in the acidic mires did the use of squeezed-water chemistry in the analyses give results similar to the use of groundwater pH. Further, we found thatSphagnum species with a similar response to the base-richness gradient had differentiated niches with respect to the water level gradient and vice versa.Sphagnum contortum andS. warnstorfii exhibiting the same demands for groundwater pH were segregated along the gradient of maximum water level. An analogous pattern was detected for acidophilous speciesSphagnum magellanicum andS. papillosum.     

Cap-binding activity of an eIF4E homolog from Leishmania

All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.     

A DECODER NMR study of backbone orientation in Nephila clavipes dragline silk under varying strain and draw rate

Using DECODER (direction exchange with correlation for orientation distribution evaluation and reconstruction) NMR, we probe the orientations of carbonyl carbons in [1-(13)C]glycine-labeled dragline silk under conditions of varying strain and fiber draw rate. A model-specific reconstruction of the molecular orientation distribution incorporating beta sheets and polyglycine II helices indicates that the structures' alignment along the fiber can be described by a pair of Gaussian distributions with full width at half-maxima of 20 and 68 degrees and approximately 45 and approximately 55% relative contributions to the signal intensity. The alignment along the fiber was found to change appreciably when the drawing tension on the fiber was relaxed in a sample drawn at 4 cm/s while little change was observed in a sample drawn at 2 cm/s. The degree of alignment along the fiber was found to increase with fiber draw rate.