Size, constant sequences, and optimal selection

Because the abundance of functional molecules in RNA sequence space has many unexplored aspects, we compared the outcome of 11 independent selections, performed using the same affinity selection protocol and contiguous randomized regions of 16, 22, 26, 50, 70, and 90 nucleotides. All affinity selections targeted the simplest isoleucine aptamer, an asymmetric internal loop. This loop should be abundant in all selections, so that it can be compared across all experiments. In some cases, two primer sets intended to favor selection of different structures have also been compared. The simplest isoleucine aptamer dominates all selections except with the shortest tract, 16 contiguous randomized nucleotides. Here the isoleucine aptamer cannot be accommodated and no other motif can be selected. Our results suggest an optimum length for selection; surprisingly, both the shortest and the longest randomized tracts make it more difficult to recover the motif. Estimated apparent initial abundances suggest that the simplest isoleucine motif was 20- to 40-fold more frequent in selection with 50- or 70-nucleotide randomized regions than with any other length. Considering primer sets, a pre-formed stable stem within fixed flanking sequences had a five-to 10-fold negative effect on apparent motif abundance at all lengths. Differing random tract lengths also determined the probable motif permutation and the most abundant helix lengths. These data support a significant but lesser role for primer sequences in the outcome of selections.     

Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.     

Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (T(H)1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particular clone to its recombinant, expressed protein. We have generated a mouse T(H)1 expression cDNA library and used protein arrays of this library to characterize the specificity and cross-reactivity of antibodies. Additionally, we have profiled the autoantibody repertoire in serum of a mouse model for systemic lupus erythematosus on these protein arrays and validated the putative autoantigens on highly sensitive protein microarrays.     

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.