DNA damage products (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyadenosines as potential biomarkers in human urine for atherosclerosis

We hypothesized that DNA damage products (5'R)-8,5'-cyclo-2'-deoxyadenosine (R-cdA) and (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) may be well-suited biomarkers of risk and diagnosis for atherosclerosis. We tested this hypothesis by measuring the levels of R-cdA and S-cdA and another product, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), in urine of atherosclerosis patients and healthy individuals using liquid chromatography-tandem mass spectrometry with isotope dilution. We showed the presence of these products at significantly greater concentrations in urine of atherosclerosis patients than in that of healthy individuals. Our data suggest that R-cdA and S-cdA can be accurately and reproducibly measured in human urine as potential biomarkers of risk and diagnosis for atherosclerosis.     

Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein

Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP^C) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP^C expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP^C expression. We show that FK506 treatment results in a profound reduction in PrP^C expression due to a defect in the translocation of PrP^C into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP^C signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP^C. However, this occurred at a later stage, after translocation of PrP^C into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP^Sc propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP^C biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases.     

Aurintricarboxylic acid structure modifications lead to reduction of inhibitory properties against virulence factor YopH and higher cytotoxicity

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability.     

Restrictase free generation of targeting vectors for disruption of complex mouse genes

Molecular cloning of targeting vectors (TgVs) is a prerequisite procedure for gene disruption in embryonic stem cells. In cases where target genes display complex features (e.g., gene overlap, alternative exon usage), TgVs must mediate deletions with very high precision to prevent unwanted effects. This is often difficult to achieve by procedures using restriction endonucleases and DNA ligases. Therefore, to prepare TgVs for inactivation of two complex genes of immunological interest: PTPRF and NWC, we employed an alternative method, which involves engineering bacterial artificial chromosomes (BACs) by inducible, plasmid encoded "Red/ET recombinase" expression system. Here, we report rapid and efficient construction of PTPRF and NWC TgVs without using restriction endonucleases.     

The influence of an electric field on growth and trace metal content in aquatic plants

It is known that both natural and artificial electric fields (EF) affect plants physiological parameters as well as germination, growth and yield. The present article describes results of a preliminary experiment on the impact of electric field on aquatic plants biogeochemistry. The objective of the present study was the assessment of the influence exerted by the electric field on growth and trace metals content of Elodea canadensis. In a laboratory experiment plants were exposed to the field intensity of 54?kV m^?1 for 7?days. The plants length was measured and the content of Fe, Mn, Ni, Pb, and Zn was determined using atomic absorption spectrometry (AAS). Results showed that the application of electric field slightly enhanced the growth of E. canadensis shoots. The content of Mn and Ni was significantly lower, and Pb and Zn significantly higher in plants exposed to the electric filed, while Fe content did not differ between control and EF treatment. This provides a rationale for further studies on biological effects of electric field in trace metal contaminated waters and application of an electrically enhanced phytoremediation.     

OGT Controls the Expression and the Glycosylation of E‐cadherin,and Affects Glycosphingolipid Structures in Human Colon Cell Lines

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongst the phenotypic changes exhibited by tumor cells, a wide range of glycosylation has been reported for colon cancer‐derived cell lines and CRC tissues. These aberrant modifications affect different aspects of glycosylation, including an increase in core fucosylation and GlcNAc branching on N‐glycans, alteration of O‐glycans, upregulated sialylation, and O‐GlcNAcylation. Although O‐GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O‐GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches on three human colon cell lines (HT29, HCT116 and CCD841CoN), it is herein reported that silencing O‐GlcNAc transferase (OGT, the sole enzyme driving O‐GlcNAcylation), only slightly affects overall N‐ and O‐glycosylation patterns. Interestingly, silencing of OGT in HT29 cells upregulates E‐cadherin (a major actor of epithelial‐to‐mesenchymal transition) and changes its glycosylation. On the other hand, OGT silencing perturbs biosynthesis of glycosphingolipids resulting in a decrease in gangliosides and an increase in globosides. Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O‐GlcNAcylation in colon cancer cells.