Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors – integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.     

Localization of human chorionic gonadotropin beta subunit transcripts in ovarian cancer tissue

Recent studies demonstrated that besides placenta and malignant trophoblastic tumors, hCG and especially its beta-subunit is secreted by a varieties of tumors of different origin. The aim of the present investigation was to determine the expression pattern of human chorionic gonadotropin gene in ovarian cancer tissue. The study included 8 patients with epithelial ovarian carcinoma. The expression and distribution of hCGbeta mRNA was assessed by in situ RT-PCR method. The semi-quantitative assessment was performed using computer image analysis. Transformation of the images into the pseudocolour scale showed a clear difference in fluorescence intensity among individual cancer cells. The intensity of ISRT-PCR products corresponding with expression level of hCGbeta demonstrated that its production by individual cancer cells is different. In all studied specimens of the ovarian carcinoma tissue, cancer cells characterized by the presence of active hCGbeta gene were found, whereas noncancerous tissue demonstrated lack of the gene expression. Thus, the study clearly shows that the expression of hCGbeta is the feature of ovarian cancer tissue.     

GSTM1 null genotype as a risk factor for anti-BPDE-DNA adduct formation in mononuclear white blood cells of coke-oven workers

The influence of the genetic deletion polymorphism of glutathione S-transferase micro 1 (GSTM1 *0/*0) on levels of anti (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-DNA) adduct in the peripheral blood lymphocyte plus monocyte fraction (LMF) of coke-oven workers was investigated. A total of 95 male Polish coke-oven workers (60% current smokers) from two different plants comprised the sample population. Polycyclic aromatic hydrocarbons (PAH) exposure was assessed by means of the individual post-shift urinary excretion of 1-pyrenol (mean +/- S.D.: 6.93 +/- 7.20 micromol/mol creatinine; 70% of the subjects exceeded the proposed biological exposure index (BEI) 2.28 micromol/mol creatinine). Anti-BPDE-DNA adduct levels were detected by high performance liquid chromatography (HPLC)/fluorescence analysis of the anti-BPDE tetrol I-1 released after acid hydrolysis of DNA samples. Genotypes were determined by polymerase chain reaction (PCR) on the genomic DNA of each subject. Coke-oven workers without active GSTM1 (GSTM1 *0/*0, 33%) had significantly higher adduct levels than those with active GSTM1 (GSTM1*1/*1 and *1/*0) (5.90 +/- 5.59 versus 3.25 +/- 2.01 adducts/10(8) bases, Mann-Whitney U-test, z = 2.53, P = 0.011), PAH exposure in the two subgroups being similar (7.06 +/- 6.83 versus 6.67 +/- 8.00 1-pyrenol micromol/mol creatinine). The highest number of GSTM1 null subjects (12/23, 39%) belonged to the quartile with the highest adduct levels (i.e., >4.67 adducts/10(8) nucleotides). That is, coke-oven workers with GSTM1 *0/*0 genotype had a significantly higher risk of having high adduct levels than individuals with active GSTM1 genotype (Fisher exact test P = 0.0355; odds ratio (OR) = 4.145, 95% CI 1.0-18.8). Multiple linear regression analysis showed that the increase in anti-BPDE-DNA adduct levels in LMF was significantly related to the high occupational exposure to PAHs (benzo[a]pyrene (BaP)) of coke-oven workers (t = 3.087, P < 0.01) and to the lack of GSTM1 activity (t = 3.512, P < 0.001), rather than to the two other confounding factors of PAH intake, i.e. charcoal-broiled meat consumption and smoking habits. In conclusion, our results indicate the clear influence of the GSTM1 detoxifying genotype on anti-BPDE-DNA adduct formation in the LMF of coke-oven workers. This is invaluable for future environmental-occupational studies using this biomarker of PAH exposure.     

Hydrolysis of fungal and plant cell walls by enzymatic complexes from cultures of Fusarium isolates with different aggressiveness to rye (Secale cereale)

The efficiency of hydrolysis of fungal (Fusarium spp.) cell wall and rye root cell wall by crude enzymatic complexes from (42-day-old) cultures of three F. culmorum isolates, a plant growth-promoting rhizosphere isolate (PGPF) DEMFc2, a deleterious rhizosphere isolate (DRMO) DEMFc5, and a pathogenic isolate DEMFc37, as well as two other, pathogenic isolates belonging to F. oxysporum and F. graminearum species was studied. In the enzymatic complexes originating from the Fusarium?spp. cultures, the activities of the following cell wall-degrading enzymes were identified: glucanases, chitinases, xylanases, endocellulases, exocellulases, pectinases, and polygalacturonases. The preparation originating from a culture of the PGPF isolate was the least efficient in plant cell wall (PCW) hydrolysis. There were no significant differences in the efficiency of PCW hydrolysis between preparations from cultures of the DRMO and the pathogenic isolates. PGPF was the most efficient in liberating reducing sugars and N-acetylglucosamine (GlcNAc) from fungal cell walls (FCW). Xylanase activities of the enzymatic complexes were strongly positively (R?>?+0.9) correlated with their efficiency in hydrolyzing PCW, whereas chitinase activities were correlated with the efficiency in FCW hydrolysis.     

2-Aminoethylphosphonate Utilization by the Cold-Adapted Geomyces pannorum P11 Strain

Cold-adapted strain of Geomyces pannorum P11 was found to mineralize of phosphorus–carbon bond-containing compound—2-aminoethylphosphonic acid (2-AEP, ciliatine). The biodegradation process proceeded in the phosphate-independent manner. Ciliatine-metabolizing enzymes' activity was detectable in cell-free extracts prepared from psychrophilic G. pannorum pregrown on 4 mM 2-AEP. Phosphonoacetaldehyde hydrolase (phosphonatase) activity in a partially purified extract was demonstrated at 10 °C.     

Carbonylated proteins accumulated as vitality decreases during long-term storage of beech (Fagus sylvatica L.) seeds

Key message

Carbonylation of proteins associated with a stress response may contribute to the lowered viability of naturally aged beech seeds, especially the desiccation tolerance-associated proteins and USP-like protein.

Abstract

Proteins are modified by a large number of reactions that involve reactive oxygen species-mediated oxidation. The direct oxidation of amino acids produces 2,4-dinitrophenylhydrazine-detectable protein products. Carbonylation is irreversible, and carbonylated proteins are marked for proteolysis or can escape degradation and form high molecular weight aggregates, which accumulate with age. Beech (Fagus sylvatica L.) seeds stored under optimal conditions for different periods of time, ranging from 2 to 13 years, were analyzed. Protein carbonylation was examined as a potential cause for the loss of viability of beech seeds, and the characteristic spots of protein carbonyls were identified. Here, we present and discuss the role of carbonylation in the proteome of beech seeds that contribute to the loss of seed viability during natural aging. The long-term storage of beech seeds is intricate because their germination capacity decreases with age and is negatively correlated with the level of protein carbonyls that accumulate in the seeds. We establish that protein synthesis, folding and degradation are the most affected biochemical traits in long-term stored beech seeds. In addition, we suggest that proteins associated with the stress response may have contributed to the lowered viability of beech seeds, especially the desiccation tolerance-associated proteins that include T-complex protein 1 and the universal stress protein (USP)-like protein, which is identified as carbonylated for first time here.     

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca²⁺ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca²⁺.     

Factors Influencing Compact–Extended Structure Equilibrium in Oligomers of Aβ1–40 Peptide—An Ion Mobility Mass Spectrometry Study

Oligomers formed by amyloid β (Aβ) peptide are widely believed to be the main neurotoxic agent in Alzheimer's disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aβ oligomers, we require systematic characterization and classification of all possible forms, facilitating rational design of the beneficial modifiers of their activity. In previous ion mobility analysis of Aβ1–40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aβ1–40 oligomers. These forms may represent two pathways of the oligomer evolution, leading either to fibrils or to off-pathway oligomers, which are potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aβ species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species.     

Induction of apoptosis in human glioma cell lines of various grades through the ROS-mediated mitochondrial pathway and caspase activation by <Emphasis Type="Italic">Rhaponticum carthamoides</Emphasis> transformed root extract

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.