X-linked inhibitor of apoptosis protein increases mitochondrial antioxidants through NF-kappaB activation

X chromosome-linked inhibitor of apoptosis protein is an endogenous inhibitor of caspases and is an important regulator of cell death. XIAP can also influence cell signaling, but downstream proteins affected are largely unknown. We show here using neuronal PC6.3 cells that XIAP increases the levels of antioxidants, particularly superoxide dismutase-2 that is localized to mitochondria. Studies using reporter constructs and NF-κB Rel-A deficient mouse embryonic fibroblasts showed that NF-κB signaling is required for the induction of Sod2 by XIAP. XIAP also reduced oxidative stress in the PC6.3 cells as shown by decreased production of reactive oxygen species. These findings disclose a novel role for XIAP in control of oxidative stress and mitochondrial antioxidants that may contribute to cell protection after various injuries.     

Mutant p53 reactivation by small molecules makes its way to the clinic

The TP53 tumor suppressor gene is mutated in many human tumors, including common types of cancer such as colon and ovarian cancer. This illustrates the key role of p53 as trigger of cell cycle arrest or cell death upon oncogenic stress. Most TP53 mutations are missense mutations that result in single amino acid substitutions in p53 and expression of high levels of dysfunctional p53 protein. Restoration of wild type p53 function in such tumor cells will induce robust cell death and allow efficient eradication of the tumor. Therapeutic targeting of mutant p53 in tumors is a rapidly developing field at the forefront of translational cancer research. Various approaches have led to the identification of small molecules that can rescue mutant p53. These include compounds that target specific p53 mutations, including PK083 and PK5174 (Y220C mutant p53) and NSC319726 (R175H mutant p53), as well as PRIMA-1 and its analog APR-246 that affect a wider range of mutant p53 proteins. APR-246 has been tested in a Phase I/II clinical trial with promising results.     

The Lonely Mouse – Single Housing Affects Serotonergic Signaling Integrity Measured by 8-OH-DPAT-Induced Hypothermia in Male Mice

Male BALB/c mice single-housed for a period of three weeks were found to respond with a more marked hypothermia to a challenge with a selective serotonergic agonist (8-OH-DPAT) than their group-housed counterparts. This effect of single housing was verified by screening a genetically heterogeneous population of male mice on a C57BL/6 background from a breeding colony. Enhanced activity of the implicated receptor (5-HT_1A) leading to an amplified hypothermic effect is strongly associated with depressive states. We therefore suggest that the 8-OH-DPAT challenge can be used to demonstrate a negative emotional state brought on by e.g. long-term single housing in male laboratory mice. The study emphasizes the importance of social housing, and demonstrates that male mice deprived of social contact respond with altered serotonergic signaling activity. Male mice not only choose social contact when given the option, as has previously been shown, but will also, when it is deprived, be negatively affected by its absence. We propose that the 8-OH-DPAT challenge constitutes a simple, but powerful, tool capable of manifesting the effect of social deprivation in laboratory mice. It potentially allows not only for an unbiased, biochemical evaluation of psychological stressors, but may also allow for determining whether the effect of these can be counteracted.     

Rapid lab-on-a-chip profiling of human gut bacteria

The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.     

Estimation of population firing rates and current source densities from laminar electrode recordings

This model study investigates the validity of methods used to interpret linear (laminar) multielectrode recordings. In computer experiments extracellular potentials from a synaptically activated population of about 1,000 pyramidal neurons are calculated using biologically realistic compartmental neuron models combined with electrostatic forward modeling. The somas of the pyramidal neurons are located in a 0.4 mm high and wide columnar cylinder, mimicking a stimulus-evoked layer-5 population in a neocortical column. Current-source density (CSD) analysis of the low-frequency part (<500 Hz) of the calculated potentials (local field potentials, LFP) based on the ‘inverse’ CSD method is, in contrast to the ‘standard’ CSD method, seen to give excellent estimates of the true underlying CSD. The high-frequency part (>750 Hz) of the potentials (multi-unit activity, MUA) is found to scale approximately as the population firing rate to the power 3/4 and to give excellent estimates of the underlying population firing rate for trial-averaged data. The MUA signal is found to decay much more sharply outside the columnar populations than the LFP.     

Informing climate models with rapid chamber measurements of forest carbon uptake

Models predicting ecosystem carbon dioxide (CO₂) exchange under future climate change rely on relatively few real‐world tests of their assumptions and outputs. Here, we demonstrate a rapid and cost‐effective method to estimate CO₂ exchange from intact vegetation patches under varying atmospheric CO₂ concentrations_. We find that net ecosystem CO₂ uptake (NEE) in a boreal forest rose linearly by 4.7 ± 0.2% of the current ambient rate for every 10 ppm CO₂ increase, with no detectable influence of foliar biomass, season, or nitrogen (N) fertilization. The lack of any clear short‐term NEE response to fertilization in such an N‐limited system is inconsistent with the instantaneous downregulation of photosynthesis formalized in many global models. Incorporating an alternative mechanism with considerable empirical support – diversion of excess carbon to storage compounds – into an existing earth system model brings the model output into closer agreement with our field measurements. A global simulation incorporating this modified model reduces a long‐standing mismatch between the modeled and observed seasonal amplitude of atmospheric CO₂. Wider application of this chamber approach would provide critical data needed to further improve modeled projections of biosphere–atmosphere CO₂ exchange in a changing climate.     

NK cell inhibitory receptor Ly-49C residues involved in MHC class I binding.

Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2D(d), as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2K(b), H-2D(b), and H-2D(d). The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2D(d) in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.     

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.     

On the 48÷80-induced secretion of tissue mast cells and its mitogenic effect on nearby cells in the intact rat

Histamine release from tissue-bound mast cells and cell proliferation in the proper mesentery in the intact rat was quantitated following in intraperitoneal injection of graded doses of compound 48/80. The dose-response curves were sigmoid-like in linear-log plots. ED50 for histamine release was 0.035-0.040 and for increased cell proliferation 0.040-0.048 microgram per g BW. The proliferative response following mast-cell secretion ceased after a period of between 48-72 h, irrespective of whether a high or a low dose of 48/80 was used. Basal on the net rate of histamine synthesis (ca. 0.45 microgram/g mesentery wet weight/h) after an initial injection of 48/80, on the extent of histamine release and the proliferative response after a repeated injection of 48/80, it is concluded that there is a lag period of at least 3 days before proliferation can be re-stimulated by renewed 48/80-induced mast-cell secretion.