Decreased emodepside sensitivity in unc-49 γ-aminobutyric acid (GABA)-receptor-deficient Caenorhabditis elegans

Emodepside, a semi-synthetic derivative of PF1022A, belongs to a new class of anthelmintic drugs, the cyclooctadepsipeptides, and shows good efficacy against macrocyclic lactone-, levamisole- or benzimidazole-resistant nematode populations. Although putative receptors for emodepside have already been discovered, its mode of action is still not fully understood. The involvement of the γ-aminobutyric acid (GABA)-receptor on the PF1022A mode of action has previously been postulated. Therefore, a possible role of the GABA-receptor, unc-49, in the mode of action of emodepside was investigated using two different Caenorhabditis elegans in vitro assays, a motility assay and a development assay. It was found that there is a clearly reduced sensitivity against emodepside of strains carrying a GABA-receptor, unc-49, loss of function mutation compared with N2 wild type C. elegans. To transfer these results from the model system to parasitic nematodes, the Toxocara canis unc-49B cDNA sequence was identified and used in a rescue experiment. The emodepside-susceptible phenotype could be fully rescued by injection of the T. canis unc-49B cDNA sequence. We believe that this is the first functional rescue of a C. elegans mutant strain with a gene from a clade III parasitic nematode. These findings, together with the earlier data on GABA-receptor binding of PF1022A, suggest that the GABA(A)-receptor UNC-49 is associated with the emodepside mode of action. However, the only partially resistant phenotype of the loss of function mutants indicates that other pathways play a more significant role.     

Morphological and immunophenotypical heterogeneity in breast cancers of young and elderly women

There is a reasonable heterogeneity in the morphological appearance and the immunohistochemical properties of distinct breast tumors. Furthermore, it is also known that cancer arising in young women have different prognosis than the ones developing in the elderly. We analyzed breast tumors of 41 young (<35 years) and 33 older women (>65 years) regarding histopathological properties and immunohistochemical reactions for ER, PgR, HER2 and Ki-67, as well as HER2 FISH. The longest diameters, thus largest available surface areas of the tumors were included in the evaluation. Different regions were marked for morphology and in all immunohistochemical reactions. The regions in the distinct tumors showing different pathological and immunohistochemical appearance were identical (p<0.001). The number of morphologically different tumor regions were more frequent in tumors developing in the young (1.82 vs. 1.48 regions/tumor), and 53.6% of tumors with heterogeneous architecture were in young vs. 39.4% in the elderly. However, regarding HER2 staining, cancers in the young patients have shown greater variability among the different tumor areas (p=0.007). The origin of tumor cells predicting prognosis remains undetermined. Whether the analysis of the expression pattern of the whole tumor is conducted or the minute regions are separately examined and averaged, the same results can be achieved. With the development of molecular techniques and accurate prognostic and treatment information rendered to samples the question may be soon answered.     

Evaluation of sulfatase-directed quinone methide traps for proteomics

Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic tools are needed. Herein, quinone methide (QM) traps directed against sulfatases are evaluated as activity-based proteomic probes (ABPPs). Starting from a p-fluoromethylphenyl sulfate scaffold, enzymatically generated QM-traps can inactivate bacterial aryl sulfatases from Pseudomonas aeruginosa and Klebsiella pneumoniae, and human steroid sulfatase. However, multiple enzyme-generated QMs form, diffuse, and non-specifically label purified enzyme. In complex proteomes, QM labeling is sulfatase-dependent but also non-specific. Thus, fluoromethylphenyl sulfates are poor ABPPs for sulfatases.     

Biochemical and pre-steady-state kinetic characterization of the hepatitis C virus RNA polymerase (NS5BDelta21, HC-J4)

Here we report a detailed characterization of the biochemical and kinetic properties of the hepatitis C virus (HCV, genotype-1b, J4 consensus) RNA-dependent RNA polymerase NS5B, by performing comprehensive RNA binding, nucleotide incorporation, and protein/protein oligomerization studies. By applying equilibrium fluorescence titrations, we determined a surprisingly high dissociation constant (K(d)) of approximately 250 nM for single-stranded as well as for partially double-stranded RNA. A detailed analysis of the nucleic acid binding mechanism using pre-steady-state techniques revealed the association reaction to be nearly diffusion controlled. It occurs in a single step with a second-order rate constant (k(on)) of 0.273 nM(-)(1) s(-)(1). The dissociation of the nucleic acid-polymerase complex is fast with a dissociation rate constant (k(off)) of 59.3 s(-)(1). With short, partially double-stranded RNAs, no nucleotide incorporation could be observed, while de novo RNA synthesis with short RNA templates showed nucleotide incorporation and end-to-end template switching events. Single-turnover, single-nucleotide incorporation studies (representing here the initiation and not processive polymerization) using dinucleotide primers revealed a very slow incorporation rate (k(pol)) of 0.0007 s(-)(1) and a K(d) of the binary enzyme-nucleic acid complex for the incoming ATP of 27.7 microM. Using dynamic laser light scattering, it could be shown for the first time that oligomerization of HCV NS5B is a dynamic and monovalent salt concentration dependent process. While NS5B is highly oligomeric at low salt concentrations, monomers were only observed at NaCl concentrations above 300 mM. Binding of short RNA substrates led to a further increase in oligomerization, whereas GTP did not show any effect on protein/protein interactions. Furthermore, nucleotide incorporation studies indicate the oligomerization state does not correlate with enzymatic activities as previously proposed.     

FTIR-microspectroscopy of prion-infected nervous tissue

The family of transmissible spongiform encephalopathies (TSE), also termed prion diseases, is a group of fatal, neurodegenerative diseases characterized by the accumulation of a misfolded protein, the disease-associated prion protein PrPSc. This glycoprotein differs in secondary structure from its normal, cellular isoform PrPC, which is physiologically expressed mostly by neurons. Scrapie is a prion disease first described in the 18th century in sheep and goats, and has been established as a model in rodents to study the pathogenesis and pathology of prion diseases. Assuming a multitude of molecular parameters change in the tissue in the course of the disease, FTIR microspectroscopy has been proposed as a valuable new method to study and identify prion-affected tissues due to its ability to detect a variety of changes in molecular structure and composition simultaneously. This paper reviews and discusses results from previous FTIR microspectroscopic studies on nervous tissue of scrapie-infected hamsters in the context of histological and molecular alterations known from conventional pathogenesis studies. In particular, data from studies reporting on disease-specific changes of protein structure characteristics, and also results of a recent study on hamster dorsal root ganglia (DRG) are discussed. These data include an illustration on how the application of a brilliant IR synchrotron light source enables the in situ investigation of localized changes in protein structure and composition in nervous cells or tissue due to PrPSc deposition, and a demonstration on how the IR spectral information can be correlated with results of complementary studies using immunohistochemistry and x-ray fluorescence techniques. Using IR microspectroscopy, some neurons exhibited a high accumulation of disease-associated prion protein evidenced by an increased amount of beta-sheet at narrow regions in or around the infected nervous cells. However, not all neurons from terminally diseased hamsters showed PrPSc deposition. Generally, the average spectral differences between all control and diseased DRG spectra are small but consistent as demonstrated by independent experiments. Along with studies on the purified misfolded prion protein, these data suggest that synchrotron FTIR microspectroscopy is capable of detecting the misfolded prion protein in situ without the necessity of immunostaining or purification procedures.     

The increased flexibility of CDR loops generated in antibodies by Congo red complexation favors antigen binding

The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the beta-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L lambda chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye-enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L lambda chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.     

Enhanced zinc consumption prevents cadmium-induced alterations in lipid metabolism in male rats

It has been investigated, based on a rat model of human exposure to cadmium (Cd), whether zinc (Zn) supplementation may prevent Cd-induced alterations in lipid metabolism. For this purpose, the concentrations of free fatty acids (FFA), phospholipids (PL), triglycerides (TG), total cholesterol (TCh), and high and low density lipoprotein cholesterol (HDL and LDL, respectively) as well as the concentrations of chosen indices of lipid peroxidation such as lipid peroxides (LPO), F₂-isoprostane (F₂-IsoP) and oxidized LDL (oxLDL) were estimated in the serum of male Wistar rats administered Cd (5 or 50 mg/l) or/and Zn (30 or 60 mg/l) in drinking water for 6 months. The exposure to 5 and 50 mg Cd/l resulted in marked alterations in the lipid status reflected in increased concentrations of FFA, TCh, LDL, LPO, F₂-IsoP and oxLDL, and decreased concentrations of PL and HDL in the serum. The concentrations of LDL, LPO, F₂-IsoP and oxLDL were more markedly enhanced at the higher Cd dosage. The supplementation with Zn during the exposure to 5 and 50 mg Cd/l entirely prevented all the Cd-induced changes in the serum concentrations of the estimated lipid compounds and indices of lipid peroxidation, except for the F₂-IsoP for which Zn provided only partial protection. Based on the results it can be concluded that Zn supplementation during exposure to Cd may have a protective effect on lipid metabolism consisting in its ability to prevent hyperlipidemia, including especially hypercholesterolemia, and to protect from lipid peroxidation. The findings seem to suggest that enhanced dietary Zn intake during Cd exposure, via preventing alterations in the body status of lipids may, at least partly, protect against some effects of Cd toxicity, including oxidative damage to the cellular membranes and atherogenic action. The paper is the first report suggesting protective impact of Zn against proatherogenic Cd action on experimental model of chronic moderate and relatively high human exposure to this toxic metal.     

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

Background

The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals.

Methodology/Principal Findings

We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst ∼8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs.

Conclusions/Significance

In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress.     

In silico approach to cisplatin toxicity. Quantum chemical studies on platinum(II)–cysteine systems

The behaviour of cisplatin in serum, and the drastic differences between the properties of this drug and its trans-isomer were the main motivations for this work. In a search for model “thiol–platin(II)” interactions, the first steps of the following reaction systems were evaluated: (1) cisplatin–thiomethanol; (2) transplatin–thiomethanol; (3) cisplatin–cysteine; and (4) transplatin–cysteine. In each case, calculations for the associative mode of reactions were performed. The electronic structure of these molecular systems was studied at the non-empirical all-electron level using density functional theory (DFT) within the Huzinaga and WTBS basis sets including polarisation Gaussian functions and full geometry optimisation. B3LYP or EPBO density functionals were applied throughout. The calculated molecular electrostatic potentials are presented graphically. Assuming that electrostatic effects are dominant, cisplatin should interact more strongly with the sulfur atom of CH₃S⁻ and deprotonated CYS-S⁻ than transplatin. This fact has been documented in the supermolecule model of the relevant interaction energies in both gas phase as well as within the solvent polarisable continuum model. The opposite relationship was observed when we compared values of energy differences between products and substrates for both isomers. The data obtained here could be applied to search for correlation between the biological activity of platinum complexes and their properties as estimated by various physico-chemical and in silico methodologies.