Quinone methide precursors protected with alkyldithiomethyl groups have been synthesized and converted into PNA conjugates. Stable in the absence of reducing agents, the electrophilic quinone methide ...is released by glutathione in concentrations typical for the cytosol. Self‐alkylation then occurs or crosslinking of RNA when hybridized with complementary strands. Fastest reactions are seen for the sterically least hindered compound.
PNA conjugates of redox‐labile quinone methide precursors can be activated by glutathione at concentrations typical for the cytosol. The quinone methide, an electrophilic short‐lived intermediate, then reacts with nucleophiles. When the PNA part is hybridized with a complementary strand of RNA, directed alkylation of the nucleobases can form crosslinks in yields up to 71 %.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Quinone methide precursors 2 and 3 were protected with a photoreactive 2-nitrobenzyl group and conjugated to peptide nucleic acids (PNA) using a Huisgen click reaction. After brief irradiation at 365 ...nm, cross-linking with complementary RNA strands started and was analyzed with an ALFexpress sequencer. When this method was used, the gel temperature had a major influence on apparent rates. Quinone methides are known to form transient as well as stable bonds with nucleotides. Although both were detected at 25 °C, analysis at 57 °C only recorded the stable types of cross-links, suggesting much slower alkylation kinetics. Linker 11 allowed us to attach quinone methides to internal positions of the PNA/RNA duplex and to capture a model of miR-20a with good efficiency.
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IJS, KILJ, NUK, PNG, UL, UM
Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and ...anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki=223nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by ...transduction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enterocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.
The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), ...and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription- translation analysis. The cloned atxA gene complemented previously characterized Tn9l7 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate
This paper describes a procedure for the cryopreservation of anchorage-dependent cells in a predefined position on microstructured glass or silicon substrates. During freezing and thawing, cells ...retain their location on the substrate, and an individual comparison and identification of cells before and after preservation are possible. To utilize this advantage, a good adherence and a high survival rate are important. It can be shown that adhesion of mouse fibroblasts (NIH-3T3) to substrate strongly influences the survival rate: 94% of cells grown for 16 h before freezing were judged to be alive after thawing. Widely spaced cells are best suited to cryopreservation on substrates. The different patterns of adhesion of cells to substrates when incubated for 1, 3, 6, and 16 h, were visualized by total internal reflection microscopy (TIRM).
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IJS, IMTLJ, KILJ, KISLJ, NUK, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Mouse fibroblasts grown on perforated Si-membranes (pore diameter ∼ 10
μm have been studied to clarify cell locomotive ability. The cell motility was microscopically monitored by a time-lapse video ...system and, simultaneously, the impedance of the growing cells was measured every 5 s. The correlations between observed cell activities and measured impedance events are discused and classified. The methods is sensitive and allows discrimination between signals arising from translocation of single cells and those arising from filopodia activities. Both cell and filopodia motion could be detected. Designs of microdevices fabricated in semiconductor technology are presented.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The Bacillus anthracis plasmid pXO1 carries genes for the synthesis of anthrax toxin. Wild-type strains produce maximal amounts of the toxin components, protective antigen (PA), lethal factor (LF), ...and edema factor (EF), only when grown in the presence of bicarbonate and CO$\sb2$. Previous work in this laboratory identified other pXO1-associated phenotypes in B. anthracis Weybridge A. These include improved growth on minimal medium, reduced frequency of sporulation, and reduced sensitivity to bacteriophages. To localize regions of pXO1 that confer these phenotypes, transposon mutagenesis utilizing pTV1 was used to generate Tn917 insertions in pXO1. Insertion mutants were isolated that (i) exhibited an increase in sensitivity to bacteriophage; (ii) produced LF and EF, but were deficient in production of PA; (iii) were deficient in production of all three toxin components; and (iv) overproduced toxin in the presence and absence of added bicarbonate and CO$\sb2$. The locations of Tn917 insertions in plasmids from the latter two types of mutants were outside of the toxin structural genes indicating that regulatory genes may have been interrupted. A gene was identified between pag and cya that encoded a trans-acting positive regulatory factor designated atxA. A region upstream of lef may be involved in negative and/or bicarbonate regulation of toxin synthesis. Restriction analysis of pXO1 from B. anthracis Weybridge A as well as from several other strains revealed restriction profiles which differed from that previously published for pXO1 from Sterne. A series of DNA-DNA hybridizations with pXO1 (Weybridge A) and pXO1 (Sterne) showed that an inversion of approximately 40 kb had occurred in the toxin-encoding region. The junctions encompassing these regions of pXO1 from Weybridge A and Sterne were cloned. Results from DNA-DNA hybridization studies with the cloned junction fragments suggested that inverted repeats of perfect or near perfect match may be present. Thus far, no differences in the plasmid-associated phenotypes have been found that can be attributed to the inversion.
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