Cadmium removal from human plasma by Cibacron Blue F3GA and thionein incorporated into polymeric microspheres

Poly(2-hydroxyethylmethacrylate–ethyleneglycoldimethacrylate) [poly(HEMA–EGDMA)] microspheres carrying Cibacron Blue F3GA and/or thionein were prepared and used for the removal of cadmium ions Cd(II) from human plasma. The poly(HEMA–EGDMA) microspheres, in the size range of 150–200 μm in diameter, were produced by a modified suspension copolymerization of HEMA and EGDMA. The reactive triazinyl dye-ligand Cibacron Blue F3GA was then covalently incorporated into the microspheres. The maximum dye incorporation was 16.5 μmol/g. Then, thionein was bound onto the Cibacron Blue F3GA-incorporated microspheres under different conditions. The maximum amount of thionein bound was 14.3 mg/g. The maximum amounts of Cd(II) ions removed from human plasma by poly(HEMA–EGDMA)–Cibacron Blue F3GA and poly(HEMA–EGDMA)–Cibacron Blue F3GA–thionein were of 17.5 mg/g and 38.0 mg/g, respectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of microspheres without significant loss in their adsorption capacity.     

Simple and efficient cell disruption of extremely small quantities of mycelium of phytopathogenic mycotoxin-producing moulds for quantitative extraction of genomic DNA

DNA was efficiently and quantitatively isolated from extremely small quantities of mycelia (0.1–10 mg) of different phytopathogenic moulds by grinding freeze-dried mycelia with glass beads and then using a commercial DNA extraction kit. The efficiency of disruption of the mycelia and the quantitative DNA extraction was proved by microscopy and the quantification of isolated DNA by real time PCR. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: German Research Foundation (DFG grant Pr 708/2). J.M. thanks the Cusanuswerk for a doctoral scholarship     

Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing

Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.     

Testing the “methanochondrion concept”: are nucleotides transported across internal membranes in Methanobacterium thermoautotrophicum?

In order to test the Methanochondrion concept, uptake of adenine nucleotides in various membrane preparations of Methanobacterium thermoautotrophicum was studied. The uptake showed properties which are in general interpreted as indicative of a transport mechanism: (i) kinetics in the time range of minutes, (ii) temperature dependence, (iii) substrate specificity and (iv) failure to remove the substrate by extensive washing.However, nucleotide transport as an interpretation of this uptake can definitely be excluded. Not only an exchange mechanism of the mitochondrial type, but also a general exchange or an uniport mechanism was ruled out. In contrast, the nucleotide uptake was shown to be actually a tight and specific binding of ADP and ATP to binding sites at the interior side of the cell membrane. This was conclusively demonstrated in protoplasts obtained from M. thermoautotrophicum cells. In these protoplasts which do not contain internal membranes also nucleotide binding was observed, but only after disruption of the plasma membrane by osmotic lysis, which leads to the exposure of binding sites.     

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.