Some Aspects of PCB Metabolism by Horseradish Cells

Transformation of polychlorinated biphenyls was studied using different strains of in vitro cultured cells of horseradish (Armoracia rusticana L.). Time and concentration dependence of this process and production of intracellular and extracellular peroxidases were measured with differentiated shooty teratoma culture K54. The yield of PCB transformation and the viability of the cells were highly dependent on PCB concentration. 100 ppm PCB totally inhibited growth of the cells, and reduced their metabolism of xenobiotics. Experiments with a peroxidase (POX) inhibitor, propylgallate, and a cytochrome P450 inhibitor, aminobenztriazole, indicated the involvement of both enzymatic systems in PCB metabolism.     

The role of defective glycosylation in congenital muscular dystrophy

The dystrophin glycoprotein complex (DGC) is an assembly of proteins spanning the sarcolemma of skeletal muscle cells. Defects in the DGC appear to play critical roles in several muscular dystrophies due to disruption of basement membrane organization. O-mannosyl oligosaccharides on α-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of α-dystroglycan to ligands (such as laminin) in the extracellular matrix and subsequent signal transmission to actin in the cytoskeleton of the muscle cell. Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome (WWS) have mutations in genes encoding glycosyltransferases needed for O-mannosyl oligosaccharide synthesis. Myodystrophic myd mice and humans with Fukuyama Congenital Muscular Dystrophy (FCMD), congenital muscular dystrophy due to defective fukutin-related protein (FKRP) and MDC1D have mutations in putative glycosyltransferases. These human congenital muscular dystrophies and the myd mouse are associated with defective glycosylation of α-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

The measurement of reactive oxygen species in human neat semen and in suspended spermatozoa: a comparison

Background  

It is generally accepted that oxidative stress is an important factor in male infertility because it may impair the physiological function of spermatozoa at the molecular level. Nevertheless, although several approaches have been reported, the imbalance between production of reactive oxygen species (ROS) and activity of the antioxidant defense system in semen is difficult to investigate and remains poorly understood.     

Rapid,nonradioactive, and quantitative method to analyze zona pellucida modifications in single mouse eggs

A rapid, nonradioactive method to monitor the ZP2 to ZP2_f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2_f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2_f conversion following A23187-induced egg activation, when compared to the commonly used radioiodination procedures. © 1994 Wiley-Liss, Inc.     

How does hydroxyl introduction influence the double helical structure: the stabilization of an altritol nucleic acid:ribonucleic acid duplex

Altritol nucleic acids (ANAs) are a promising new tool in the development of artificial small interfering ribonucleic acids (siRNAs) for therapeutical applications. To mimic the siRNA:messenger RNA (mRNA) interactions, the crystal structure of the ANA:RNA construct a(CCGUAAUGCC-P):r(GGCAUUACGG) was determined to 1.96?? resolution which revealed the hybrid to form an A-type helix. As this A-form is a major requirement in the RNAi process, this crystal structure confirms the potential of altritol-modified siRNAs. Moreover, in the ANA strands, a new type of intrastrand interactions was found between the O2' hydroxyl group of one residue and the sugar ring O4' atom of the next residue. These interactions were further investigated by quantum chemical methods. Besides hydration effects, these intrastrand hydrogen bonds may also contribute to the stability of ANA:RNA duplexes.     

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

TRAIL induces apoptosis but not necroptosis in colorectal and pancreatic cancer cells preferentially via the TRAIL-R2/DR5 receptor

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that can trigger apoptosis in many types of human cancer cells via engagement of its two pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). TRAIL can also activate several other signaling pathways such as activation of stress kinases, canonical NF-κB signaling and necroptosis. Though both receptors are ubiquitously expressed, their relative participation in TRAIL-induced signaling is still largely unknown. To analyze TRAIL receptor-specific signaling, we prepared Strep-tagged, trimerized variants of recombinant human TRAIL with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, we examined the contribution of individual pro-apoptotic receptors to TRAIL-induced signaling pathways. We found that in TRAIL-resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeds comparably via both DR4- and DR5-activated signaling. TRAIL-induced apoptosis, enhanced by the inhibitor of the Bcl-2 family ABT-737, or by the translation inhibitor homoharringtonine, proceeded in both cell lines predominantly via the DR5 receptor. ShRNA-mediated downregulation of DR4 or DR5 receptors in HT-29 cells also pointed to a stronger contribution of DR5 in TRAIL-induced apoptosis. In contrast to apoptosis, necroptotic signaling was activated similarly by both DR4- or DR5-specific ligands. Activation of auxiliary signaling pathways involving NF-κB or stress kinases proceeded under apoptotic conditions mainly in a DR5-dependent manner, while these signaling pathways were during necroptosis similarly activated by either of these ligands. Our study provides the first systematic insight into DR4 ?/DR5-specific signaling in colorectal and pancreatic cancer cells.     

Regulation of growth,nutritive, phytochemical and antioxidant potential of cultivated <Emphasis Type="Italic">Drimiopsis maculata</Emphasis> in response to biostimulant (vermicompost leachate,VCL) application

The effect of vermicompost leachate (VCL, low-cost biostimulant) on the growth, elemental (macro and micro-nutrients) and phytochemical content as well as the antioxidant potential of Drimiopsis maculata was evaluated. Three dilutions (1:5; 1:10 and 1:20) of VCL were tested and the cultivation lasted for 3 months. In addition to the recorded growth parameters, dried and ground plant materials (leaves and bulbs) were evaluated for nutrients, phenolic acids and antioxidant capacity. Vermicompost leachate application enhanced the growth of D. maculata, particularly, the leaves (VCL 1:10) and bulbs (VCL 1:20) which were significantly bigger than the controls. Apart from the concentration of phosphorus which was significantly lower in the leaves of VCL (1:20)-treated plants, the quantity of all four macro-nutrients analysed were similar with and without VCL. Similar observations were also demonstrated in the majority of quantified micro-nutrients in D. maculata. Relative to the control, VCL-treated plants had higher concentrations of the 10 phenolic acids quantified in the leaves. However, the majority of the quantified phenolic acids were not significantly enhanced in bulbs. Antioxidant activity of D. maculata extracts was generally higher in leaves than in the bulbs. The leaf extract from VCL (1:10 and 1:20)-treated plants exhibited lower oxygen radical absorbance capacity (ORAC) when compared to the control. However, bulbs from VCL (1:5) treatment had significantly higher ORAC than the control. From a conservational perspective, the current findings provided insight on viable approaches useful for mitigating challenges associated with over-harvesting of highly utilized but slow-growing plant species.