Hydroquinone inhibits PMA-induced activation of NFkappaB in primary human CD19+ B lymphocytes

Hydroquinone (HQ), a reactive metabolite of benzene, is known to inhibit mitogen-stimulated activation of both T and B lymphocytes. Despite extensive study, the underlying mechanism for the immunotoxicity of the HQ is not clear. We have previously demonstrated that 1 micromol/L HQ inhibits TNF-induced activation of NFkappaB in CD4+ T cells, resulting in decreased IL-2 production. NFkappaB, known to be important in T lymphocytes, also plays a critical role in normal B cell development and activation. We therefore hypothesized that alterations in NFkappaB might be involved in HQ-induced B cell immunosuppression as well. In this study, we demonstrate that 1-10 micromol/L HQ inhibits PMA/ionomycin-induced activation of NFkappaB in primary human CD19+ B cells. Inhibition of NFkappaB is accompanied by a dose-dependent decrease in PMA-stimulated production of TNF with no corresponding loss in viability or increased apoptosis. HQ also does not appear to alter NFkappaB directly, as preincubation of B cell nuclear extracts with HQ does not diminish DNA binding activity of this protein. In contrast to T cells, inhibition of NFkappaB by HQ in B cells is not reversible after 72 h in culture, suggesting a long-term functional suppression. These data support our original findings in T cells and indicate that NFkappaB is particularly susceptible to inhibition by HQ. We further hypothesize that inhibition of NFkappaB in lymphocytes, and perhaps other cell types as well, may play a significant role in the observed toxicity of HQ.     

Downregulation of calcineurin activity in cervical carcinoma

Background  

Calcineurin (CaN) is an important serine-threonine phosphatase (PP2B), which plays a crucial role in calcium-calmodulin mediated signal transduction events. Calcineurin has been implicated in pathogenesis of various diseases cardiac hypertrophy, diabetic neuropathy and Alzheimer's, however its role in neoplasia remains unclear.     

Expression microarray hybridization kinetics depend on length of the immobilized DNA but are independent of immobilization substrate

Expression microarrays are often constructed by the immobilization of PCR products on two-dimensional modified glass slides or on three-dimensional microporous substrates. In this study we investigate whether the length of the immobilized species and the substrate choice influence hybridization dynamics. Using a simple bimolecular mass action controlled model to describe hybridization, we observed that the extent of hybridization and the initial velocities were directly dependent on the length of the immobilized species. An inflection point was noted at a length of 712 bases, above which the influence of length on hybridization rate decreased. Interestingly, we observed no differences in these parameters whether hybridization occurred on a two- or three-dimensional surface. Furthermore, the affinity of the solution phase labeled species for the immobilized species was identical for all arrayed lengths on both surfaces. These data indicate a similar interaction of the noncovalently immobilized species with either surface. Finally, we have determined that competitive hybridization on expression microarrays is nonlinear with respect to time and concentration of competitor. This observation is critical for analysis of expression array data.     

Probing structural changes in the α and β domains of copper- and silver-substituted metallothionein by emission spectroscopy and electrospray ionization mass spectrometry

Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted α and β domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag(I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag(I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted α and β domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag(I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted α and β fragments than when the Ag(I)-MT species were formed from the apo-α and apo-β fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag(I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.     

Benefits to shorebirds from invasion of a non-native shellfish

Introductions of non-native species are seen as major threats to ecosystem function and biodiversity. However, invasions of aquatic habitats by non-native species are known to benefit generalist consumers that exhibit dietary switches and prey upon the exotic species in addition to or in preference to native ones. There is, however, little knowledge concerning the population-level implications of such dietary changes. Here, we show that the introduction of the Manila clam Tapes philippinarum into European coastal waters has presented the Eurasian oystercatcher Haematopus ostralegus ostralegus with a new food resource and resulted in a previously unknown predator-prey interaction between these species. We demonstrate, with an individuals-based simulation model, that the presence of this non-native shellfish, even at the current low density, has reduced the predicted over-winter mortality of oystercatchers at one recently invaded site. Further increases in clam population density are predicted to have even more pronounced effects on the density dependence of oystercatcher over-winter mortality. These results suggest that if the Manila clam were to spread around European coastal waters, a process which is likely to be facilitated by global warming, this could have considerable benefits for many shellfish-eating shorebird populations.     

Structural properties of metal-free apometallothioneins

The metalated forms of metallothionein are well studied (particularly Zn-MT, Cu-MT and Cd-MT), but almost nothing is known about the chemical and structural properties of apometallothioneins despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn(7)-MT and apo-MT at pH 7 exhibit the same charge state distribution, indicating that apo-MT is tightly folded like the metallated protein, whereas apo-MT at pH 3 exhibits a charge state spectrum associated with unfolding or denaturation. Benzoquinone was used to modify the cysteines in the β-MT (9Bq), and α-MT (11Bq) fragments, and the full βα-MT (20Bq) protein. ESI-MS showed that the overall volume and, therefore, the extent of folding for the modified proteins is similar to that of Zn-MT. Molecular modeling using MM3-MD methods provided the volume of each modified protein. The volumes of the partially modified proteins follow the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chemical stress. The data suggest that the structure of apo-βα-MT is more organized than previously considered.