Changes in the binding of copper in the plasma of molybdenum supplemented rats

After incubating plasma of Mo-supplemented rats (Mo-plasma) with ⁶⁴Cu only part of it could be removed by dialysis against EDTA or histidine or by treatment with dithiocarbamate; this nondialyzable Cu was shown to be bound to albumin. The maximal amount of ⁶⁴Cu bound this way equaled the Mo-induced increase in total plasma Cu. After addition of stable Cu dialysis of Mo-plasma against a histidine solution showed that no extra Cu became tightly bound, suggesting that the ⁶⁴Cu binding was due to an exchange between added ⁶⁴Cu and stable Cu already present. Incubating Mo-plasma with Hg compounds prevented ⁶⁴Cu binding and released stable Cu, indicating that Cu in Mo-plasma was sulfhydryl bound. Part of the Mo in Mo-plasma was freely dialyzable. The remaining part was shown to be SH bound as well. The estimated atomic ratio of SH-bound Cu and Mo was unity. Molybdenum increased the number of SH groups in plasma, and for each Cu atom at least one SH group was calculated to be present.     

Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1 ^−/−‐ and Tlr8 ^−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.     

Kinetic models for detection of toxicity in a microbial fuel cell based biosensor

Currently available models describing microbial fuel cell (MFC) polarization curves, do not describe the effect of the presence of toxic components. A bioelectrochemical model combined with enzyme inhibition kinetics, that describes the polarization curve of an MFC-based biosensor, was modified to describe four types of toxicity. To get a stable and sensitive sensor, the overpotential has to be controlled. Simulations with the four modified models were performed to predict the overpotential that gives the most sensitive sensor. These simulations were based on data and parameter values from experimental results under non-toxic conditions. Given the parameter values from experimental results, controlling the overpotential at 250 mV leads to a sensor that is most sensitive to components that influence the whole bacterial metabolism or that influence the substrate affinity constant (Km). Controlling the overpotential at 105 mV is the most sensitive setting for components influencing the ratio of biochemical over electrochemical reaction rate constants (K1), while an overpotential of 76 mV gives the most sensitive setting for components that influence the ratio of the forward over backward biochemical rate constants (K2). The sensitivity of the biosensor was also analyzed for robustness against changes in the model parameters other than toxicity. As an example, the tradeoff between sensitivity and robustness for the model describing changes on K1 (IK1) is presented. The biosensor is sensitive for toxic components and robust for changes in model parameter K2 when overpotential is controlled between 118 and 140 mV under the simulated conditions.