Influence of salinity and dissolved organic carbon on acute Cu toxicity to the rotifer Brachionus plicatilis.

Acute copper (Cu) toxicity tests (48-h LC50) using the euryhaline rotifer Brachionus plicatilis were performed to assess the effects of salinity (3, 16, 30 ppt) and dissolved organic carbon (DOC, ∼ 1.1, ∼ 3.1, ∼ 4.9, ∼ 13.6 mg C L(-1)) on Cu bioavailability. Total Cu was measured using anodic stripping voltammetry, and free Cu(2+) was measured using ion-selective electrodes. There was a protective effect of salinity observed in all but the highest DOC concentrations; at all other DOC concentrations the LC50 value was significantly higher at 30 ppt than at 3 ppt. At all salinities, DOC complexation significantly reduced Cu toxicity. At higher concentrations of DOC the protective effect increased, but the increase was less than expected from a linear extrapolation of the trend observed at lower concentrations, and the deviation from linearity was greatest at the highest salinity. Light-scattering data indicated that salt induced colloid formation of DOC could be occurring under these conditions, thereby decreasing the number of available reactive sites to complex Cu. When measurements of free Cu across DOC concentrations at each individual salinity were compared, values were very similar, even though the total Cu LC50 values and DOC concentrations varied considerably. Furthermore, measured free Cu values and predicted model values were comparable, highlighting the important link between the concentration of bioavailable free Cu and Cu toxicity.

Cofractionation of HMGB proteins with histone dimers.

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A-H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein-protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry.

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.

The oxidised histone octamer does not form a H3 disulphide bond.

A H3 dimer band is produced when purified native histone octamers are run on an SDS-PAGE gel in a beta-mercaptoethanol-free environment. To investigate this, native histone octamer crystals, derived from chicken erythrocytes, and of structure (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A'), were grown in 2 M KCl, 1.35 M potassium phosphates and 250-350 microM of the oxidising agent S-nitrosoglutathione, pH 6.9. X-ray diffraction data were acquired to 2.10 A resolution, yielding a structure with an Rwork value of 18.6% and an Rfree of 22.5%. The space group is P6(5), the asymmetric unit of which contains one complete octamer. Compared to the 1.90 A resolution, unoxidised native histone octamer structure, the crystals show a reduction of 2.5% in the c-axis of the unit cell, and free-energy calculations reveal that the H3-H3' dimer interface in the latter has become thermodynamically stable, in contrast to the former. Although the inter-sulphur distance of the two H3 cysteines in the oxidised native histone octamer has reduced to 6 A from the 7 A of the unoxidised form, analysis of the hydrogen bonds that constitute the (H4-H3)-(H3'-H4') tetramer indicates that the formation of a disulphide bond in the H3-H3' dimer interface is incompatible with stable tetramer formation. The biochemical and biophysical evidence, taken as a whole, is indicative of crystals that have a stable H3-H3' dimer interface, possibly extending to the interface within an isolated H3-H3' dimer, observed in SDS-PAGE gels.

High-resolution structure of the native histone octamer.

Crystals of native histone octamers (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A') from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 A resolution, yielding a structure with an R(work) value of 18.7% and an Rfree of 22.2%. The crystal space group is P6(5), the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A'-H3-H4 molecular cluster (also H2A-H3'-H4'), the H4-H2B' interaction (also H4'-H2B) and the H2A'-H4 beta-sheet interaction (also H2A-H4'). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.

Histone structures: targets for modifications by molecular assemblies.

The core histone proteins contain modification sites that are key elements in the regulation of the cell cycle, DNA replication and repair with histone assembly, control of gene expression, and transcriptional elongation. Much work has been done on the various molecular assemblies that remodel nucleosomes, methylate, ubiquitinate, and cause ADP-ribosylation of histones, and acetylate and phosphorylate core histone tails. The core histones are the final targets of the enzymes in the molecular assemblies. What structural changes in the histones are correlated with these modifications? This paper considers the high-resolution structure of the histone octamer and stresses the importance of histone docking sequences in the binding of the two (H2A-H2B) dimers to the (H3-H4)(2) tetramer. There is an extensive acid-base area of interaction between histone octamers in crystals at high salt, which may have implications for nucleosome remodeling. We show that there are regions of high alpha-helix probability in all core histone N-terminal tails in regions where lysine acetylation occurs. There are also consensus sequences spanning up to eight amino acid residues between some histone tail regions. Circular dichroism studies using synchrotron radiation at wavelengths as low as 130 nm are promising for the accurate measurement of changes of histone secondary structure related to function.

Precautions in the use of 110mAg as a tracer of silver metabolism in ecotoxicology: preferential bioconcentration of 109Cd by trout gills after 110mAg exposure.

An often overlooked problem in the use of radiotracers is the possibility of isotopic contamination. Commercially available silver 110mAg was used to study silver uptake and depuration in rainbow trout and European eel. Quality control by means of comparative gamma and beta counting brought our attention to a contamination of the 110mAg stock with 109Cd, which could be seen only because the 109Cd was markedly bioconcentrated by trout gills. The contamination could not be detected in eel gills or in other tissues of both species. The difference between trout and eel gill structure and function is the probable explanation for the marked difference in 109Cd accumulation. This contamination was identified as 109Cd by gamma spectroscopy and its origin by transmutation of natural silver as a result of neutron activation is described. Failure to recognize this contamination problem would have resulted in serious misinterpretation of the data set. Guidance for avoiding this problem is given.