Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis

A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm × 75 μm I.D.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25°C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10⁵-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.     

New proteolytic enzymes in yeast

Yeast mutants lacking three proteolytic enzymes—proteinase B, carboxypeptidase Y, and carboxypeptidase S—have been constructed. Search for new proteolytic activities in these mutants with the aid of chromogenic peptide substrates developed for serum proteinases led to the detection of new proteolytic activities, active in the neutral pH range. Sephadex chromatography of a 100,000g supernate of mutant extracts, tests against four different substrates, and partial characterization of their sensitivity to various inhibitors indicate multiple activities. Two activities, called proteinase M and proteinase P, were found in the sedimentable membranous fraction of mutant extracts.     

Dose- and Wavelength-Dependent Oxidation of Crystallins by UV Light-Selective Recognition and Degradation by the 20S Proteasome

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of -, β_L-, and β_H-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.     

Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron-repressible outer membrane polypeptide of 65 000 Da and pesticin sensitivity

Iron-repressible outer membrane proteins (Irp) and siderophore production of Yersinia enterocolitica, serotype 08, were subjected to analysis. Here four Irps of apparent molecular weights of 62000, 65000, 74000 and 75000 could be identified which were expressed constitutively by a fur mutant. Production of a novel catechol-containing siderophore (denoted yersiniabactin) was detected by siderophore-indicator agar (chrome azurol S) and feeding experiments. Growth support by yersiniabactin under iron-restricted conditions was TonB- and Irp65-dependent and correlated with pesticin-sensitivity of Yersinia enterocolitica and Escherichia coliø. From these results we conclude that Irp65 of Y. enterocolitica functions as yersiniabactin receptor (FyuA) and as pesticin receptor. By immunoblotting using rabbit antibodies against Irp65 and chrome azurol S-agar, we were able to demonstrate that all tested mouse-lethai Y. enterocoiitica and Yersinia pseudotuberculosis strains of different serotypes express siderophores and Irp65. Moreover, the anti-lrp65 rabbit serum did not cross-react with the known iron-repressible high-molecular-weight proteins (HMWPs). Evidently, the mouse lethality trait in enteropathogenic Yersinia spp. is closely associated with a novel iron-uptake system, comprising the production of a siderophore and a siderophore receptor of apparent molecular mass 65 000 Da.     

Ultrastructural,cytochemical, and immunocytochemical characterization of haemocytes of the hard tick Ixodes ricinus (Acari; Chelicerata)

Haemocytes of the hard tick Ixodes ricinus were characterized on the basis of their ultrastructure, their ability to ingest foreign material, and to produce or store molecules of the immune defence. Distinction was made between types of haemocytes according to the absence or presence of granular inclusions, shape and size of the lysosomal compartment or the rough endoplasmic reticulum, and ultrastructural and functional similarity to the corresponding haemocytes of insects. Three types of haemocytes were found in adult ticks: plasmatocytes and type-I and type-II granular haemocytes, respectively. The precipitated reaction product of acid phosphatase activity revealed the shape of the lysosomal compartment. The additional injection of particulate materials into the haemocoel further revealed the endocytic activity of the haemocytes. The lysozyme-like immunoreactivity of the haemocytes suggests bactericidal potential. Detection of immunoreactivity in haemocytes to a 25 kDa antigenic protein involved in cuticle formation further suggests their involvement in wound healing and encapsulation.     

Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis

We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.     

Soil fertility increases with plant species diversity in a long-term biodiversity experiment

Most explanations for the positive effect of plant species diversity on productivity have focused on the efficiency of resource use, implicitly assuming that resource supply is constant. To test this assumption, we grew seedlings of Echinacea purpurea in soil collected beneath 10-year-old, experimental plant communities containing one, two, four, eight, or 16 native grassland species. The results of this greenhouse bioassay challenge the assumption of constant resource supply; we found that bioassay seedlings grown in soil collected from experimental communities containing 16 plant species produced 70% more biomass than seedlings grown in soil collected beneath monocultures. This increase was likely attributable to greater soil N availability, which had increased in higher diversity communities over the 10-year-duration of the experiment. In a distinction akin to the selection/complementarity partition commonly made in studies of diversity and productivity, we further determined whether the additive effects of functional groups or the interactive effects of functional groups explained the increase in fertility with diversity. The increase in bioassay seedling biomass with diversity was largely explained by a concomitant increase in N-fixer, C4 grass, forb, and C3 grass biomass with diversity, suggesting that the additive effects of these four functional groups at higher diversity contributed to enhance N availability and retention. Nevertheless, diversity still explained a significant amount of the residual variation in bioassay seedling biomass after functional group biomass was included in a multiple regression, suggesting that interactions also increased fertility in diverse communities. Our results suggest a mechanism, the fertility effect, by which increased plant species diversity may increase community productivity over time by increasing the supply of nutrients via both greater inputs and greater retention.