High diversity of ankA sequences of Anaplasma phagocytophilum among Ixodes ricinus ticks in Germany

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.     

Heat shock protein 70 associations with myelin basic protein and proteolipid protein in multiple sclerosis brains

Heat shock proteins (hsp) are known to facilitate the generation of specific immune responses by chaperoning proteins and peptides involved in T cell activation. Hsp have been shown to be strikingly elevated in multiple sclerosis (MS) lesions. The unique chaperonin properties of hsp70 have allowed identification of immunogenic proteins bound to it by the ex vivo demonstration of hsp associations with proteins implicated in the immune response. We have investigated the association of hsp70 with myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) in MS and control brain tissue. In co-immunoprecipitation experiments, in all samples of MS brains examined (n = 3), but not control brain tissue (n = 3), direct association of MBP with hsp70, but not with hsp90, was found. In some MS brain samples, association between PLP and hsp70 was also seen. In similar co-immunoprecipitation experiments on brain tissue obtained from mice with experimental autoimmune encephalomyelitis (n = 5) induced by immunization with PLP peptide, specific association of hsp70 with PLP and MBP was found. Using surface plasmon resonance we demonstrated specific binding of hsp70 with MBP in vitro. Analysis of the amounts of MBP bound to hsp70 yielded a molecular ratio of MBP binding to hsp70 at 6.5:1. MBP complexed with hsp70 was taken up at significantly higher rates by antigen-presenting cells than MBP alone and enhanced MBP-specific immune responses. These results indicate that hsp70 specifically associates with MBP in MS brain tissue. This association might be relevant to the enhanced immune recognition of MBP in MS.     

Blood serum levels of vascular cell adhesion molecule (sVCAM-1), intercellular adhesion molecule (sICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) in diabetic retinopathy

BACKGROUND: Interaction between cells via intimate cell-cell contact is facilitated by a cell surface molecules, termed adhesion molecules. The aim of the study was to evaluate the blood serum concentration of soluble forms of vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) in patients with type 1 diabetes mellitus without and with diabetic retinopathy. MATERIALS AND METHODS: The study was performed in 75 patients with type 1 diabetes mellitus, 35 without retinopathy (group 1) and 40 with retinopathy (group 2). Soluble forms of VCAM-1, ICAM-1 and ELAM-1 were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum concentration of sICAM-1 and sELAM-1 were significantly elevated and the concentration sVCAM-1 was elevated but not significantly in diabetic patients when compared with control subjects. There was a significant difference in VCAM-1 concentrations between the control group and group 2 (965.9 +/- 229.0 vs. 1283.7 +/- 387.6 ng/ml, p < 0.05) and between group 1 and group 2 (1115.0 +/- 285.5 vs. 1283.7 +/- 387.6 ng/ml, p < 0.05). There were significant differences in sICAM-1 concentrations between the control group and group 1 (p < 0.05) and between the control group and group 2 (p < 0.05). Where was no significant difference in sICAM-1 concentration between group 1 and 2 (405.2 +/- 135.9 vs. 443.1 +/- 112.7 ng/ml, p = 0.08). ELAM-1 concentration was significantly elevated in group 2 (120.5 +/- 49.3 ng/ml) when compared with the control group (51.7 +/- 18.1 ng/ml, p < 0.005) and with group 1 (81.2 +/- 27.7 ng/ml, p < 0.05). CONCLUSIONS: The correlations found between sVCAM-1, sICAM-1 and sELAM-1 and the presence of retinopathy suggest that cellular adhesion and neovascularization may be linked processes.     

Zinc is a voltage-dependent blocker of native and heterologously expressed epithelial Na+ channels

Zn(2+) (1-1,000 microM) applied to the apical side of polarized A6 epithelia inhibits Na(+) transport, as reflected in short-circuit current and conductance measurements. The Menten equilibrium constant for Zn(2+) inhibition was 45 microM. Varying the apical Na(+) concentration, we determined the equilibrium constant of the short-circuit current saturation (34.9 mM) and showed that Zn(2+) inhibition is non-competitive. A similar effect was observed in Xenopus oocytes expressing alphabetagammarENaC (alpha-, beta-, and gamma-subunits of the rat epithelial Na(+) channel) in the concentration range of 1-10 microM Zn(2+), while at 100 microM Zn(2+) exerted a stimulatory effect. The analysis of the voltage dependence of the steady-state conductance revealed that the inhibitory effect of Zn(2+) was due mainly to a direct pore block and not to a change in surface potential. The equivalent gating charge of ENaC, emerging from these data, was 0.79 elementary charges, and was not influenced by Zn(2+). The stimulatory effect of high Zn(2+) concentrations could be reproduced by intra-oocyte injection of Zn(2+) (approximately 10 microM), which had no direct effect on the amiloride-sensitive conductance, but switched the effect of extracellular Zn(2+) from inhibition to activation.     

Integrating polyurethane culture substrates into poly(dimethylsiloxane) microdevices

Poly(dimethylsiloxane) (PDMS)-based microdevices have enabled rapid, high-throughput assessment of cellular response to precisely controlled microenvironmental stimuli, including chemical, matrix and mechanical factors. However, the use of PDMS as a culture substrate precludes long-term culture and may significantly impact cell response. Here we describe a method to integrate polyurethane (PU), a well-studied and clinically relevant biomaterial, into the PDMS multilayer microfabrication process, enabling the exploration of long-term cellular response on alternative substrates in microdevices. To demonstrate the utility of these hybrid microdevices for cell culture, we compared initial cell adhesion, cell spreading, and maintenance of protein patterns on PU and PDMS substrates. Initial cell adhesion and cell spreading after three days were comparable between collagen-coated PDMS and PU substrates (with or without collagen coating), but significantly lower on native PDMS substrates. However, for longer culture durations (≥6 days), cell spreading and protein adhesion on PU substrates was significantly better than that on PDMS substrates, and comparable to that on tissue culture-treated polystyrene. Thus, the use of a generic polyurethane substrate in microdevices enables longer-term cell culture than is possible with PDMS substrates. More generally, this technique can improve the impact and applicability of microdevice-based research by facilitating the use of alternate, relevant biomaterials while maintaining the advantages of using PDMS for microdevice fabrication.     

High-resolution whole-organ mapping with SNPs and its significance to early events of carcinogenesis

We attempted to identify deleted segments in two model tumor suppressor gene loci on chromosomes 13q14 and 17p13 that were associated with clonal expansion of in situ bladder preneoplasia using single nucleotide polymorphisms (SNPs)-based whole-organ histologic and genetic mapping. For mapping with SNPs, the sequence-based maps spanning approximately 27 and 5 Mb centered around RB1 and p53, respectively, were assembled. The integrated gene and SNP maps of the regions were used to select 661 and 960 SNPs, which were genotyped by pyrosequencing. Genotyping of SNPs was performed on DNA samples corresponding to histologic maps of the entire bladder mucosa in human cystectomy specimens with invasive urothelial carcinoma. By using this approach, we have identified deleted regions associated with clonal expansion of intraurothelial neoplasia; which ranged from 0.001 to 4.3 Mb (average 0.67 Mb) and formed clusters of discontinuous deleted segments. The high resolution of such maps is a prerequisite for future positional targeting of genes involved in early phases of bladder neoplasia. This approach also permits analysis of the overall genomic landscape of the involved region and discloses that a unique composition of noncoding DNA characterized by a high concentration of repetitive sequences may predispose to deletions.     

Functional activity of the carboxyl-terminally extended oxytocin precursor Peptide during cardiac differentiation of embryonic stem cells

The hypothalamic post-translational processing of oxytocin (OT)-neurophysin precursor involves the formation of C-terminally extended OT forms (OT-X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT-X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor-mediated cardiomyogenic action of OT-Gly-Lys-Arg (OT-GKR). To obtain further insight into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing the OT-GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT-GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT-GKR/EGFP-expressing embryoid bodies and elevated expression of GATA-4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT-GKR on ES cell-derived cardiomyogenesis. Furthermore, patch-clamp experiments demonstrated induction of ventricular phenotypes in OT-GKR/EGFP-transfected and in OT-GKR-treated cardiomyocytes. Increased connexin 43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.     

Vitamin C modulates DNA damage induced by hydrogen peroxide in human colorectal adenocarcinoma cell lines (HT29) estimated by comet assay in vitro

Introduction

Cancer cells, compared to normal cells, are under increased oxidative stress associated with oncogenic transformation, alterations in metabolic activity, and increased generation of reactive oxygen species.

Material and methods

We investigated the ability of vitamin C to reduce the damage induced by hydrogen peroxide, in human colorectal adenocarcinoma cells in vitro by the comet assay. Additionally, we measured the kinetics and efficacy of the repair of DNA damage after incubation with vitamin C in the presence of H₂O₂.

Results

The obtained results showed that 1 h pre-incubation with vitamin C and exposure to H₂O₂ for the last 10 min of incubation caused a statistically significant (p < 0.05) increase in DNA migration in comet tails in all experimental series. For the 10 µM, 25 µM, 50 µM, 100 µM vitamin C concentrations the levels of DNA damage were as follows: 18.6%, 21.1%, 25.3% and 27.2%, respectively, as compared to the untreated cells (3.26%). However, in comparison with H₂O₂ alone (29.1%), we observed a statistically significant (p < 0.05) decrease of the genotoxic effect in HT29 cells induced by H₂O₂ for the two lowest of concentrations of vitamin C: 10 µM and 25 µM. The HT29 cells were able to achieve effective repair of the damaged DNA within 60 and 120 min after incubation with the tested compounds. All the values obtained in the test were statistically significant (p < 0.05).

Conclusions

Vitamin C caused a weaker DNA damaging effect of hydrogen peroxide and positively influences the level of oxidative DNA damage in HT29 cells (decrease ∼ 30%). We noted that DNA damage was effectively repaired during 120 min postincubation in the tested cells and that oxidative damage was the major type of damage.     

The function of type I interferons in antimicrobial immunity

Type I interferons (IFN-alpha and IFN-beta) were originally described as potent antiviral substances, which are produced upon infection of animal cells with viruses. Despite a large body of literature that has accumulated during the past 25 years, their regulatory function in the immune system is still much less appreciated. Recent studies have highlighted the production of type I IFNs, their function in the immune response to infectious agents and the target cells of these interferons. Type I IFNs clearly affect the release of proinflammatory cytokines or nitric oxide by dendritic cells and macrophages, the capacity of type II interferon (IFN-gamma) to activate phagocytes, the differentiation of T helper cells and the innate control of non-viral pathogens.