Lipid Peroxidation and Antioxidant System in the Tumor and in the Blood of Patients with Nasopharyngeal Carcinoma

Reactive oxygen species play a key role in cancer development by inducing and maintaining the oncogenic phenotypes of cancer cells. In this study, we examined lipid peroxidation and antioxidant enzymes activities in the blood and in the tumor of nasopharyngeal carcinoma patients. Plasma malondialdehyde, conjugated dienes, erythrocytes catalase, and superoxide dismutase activities have been measured in 30 untreated nasopharyngeal carcinoma patients and 30 controls on one hand. On the other hand, tumor malondialdehyde level, catalase, and superoxide dismutase activities have been measured in five nasopharyngeal carcinoma patients and compared with four controls. The lipid peroxidation was confirmed in the plasma by the high levels of malondialdehyde and conjugated dienes (p?<?0.001, respectively). Additionally, significantly higher concentrations of malondialdehyde were found in biopsies compared to the control group (p?<?0.001). In erythrocytes, superoxide dismutase activity was higher in patients than in controls (p?<?0.05), while it was unchanged in the tumor (p?>?0.05). Both erythrocytes and tumor catalase activities were significantly lower in patients than in controls (p?<?0.001, respectively). Statistical studies have shown a positive correlation between malondialdehyde level and IgA antibodies level against Epstein–Barr virus capsid antigen (p?<?0.05). In conclusion, we reported the presence of an oxidative stress in the blood and in the biopsies of nasopharyngeal carcinoma patients where Epstein–Barr virus seems to play a role.     

Acute graft-vs.-host disease correlates with the disparity for the PECAM-1 S536N polymorphism only in the HLA-B44-like positive Tunisian recipients of HSCs

GVHD is the major cause of mortality after HLA-identical HSCT. Such complication has been widely linked to donor/recipient disparity for minor histocompatibility antigens (MiHAgs). PECAM-1 is one of potential human MiHAgs but its effect on the GVHD occurrence remains not clear. In order to examine such association in the Tunisian cohort of HSCs recipients, we performed a retrospective study on patients who undergone HLA-identical HSCT between 2000 and 2009. Genotyping of the three selected PECAM-1 polymorphisms (rs668, rs12953 and rs1131012) was performed with SSP-PCR method. Univariate analyses showed that grades II-IV acute GVHD were considerably linked to the non-identity for rs12953 only in HLA-B44-like positive patients (p = 0.010, OR = 10.000). Multivariate analysis for chronic GVHD showed that this outcome may be affected only by the adulthood and the conditioning regimen. Our findings support the previously reported data suggesting a significant association between the PECAM-1 disparity and the risk of acute GVHD.     

Genomic aberrations in lung adenocarcinoma in never smokers

Background

Lung cancer in never smokers would rank as the seventh most common cause of cancer death worldwide.

Methods and Findings

We performed high-resolution array comparative genomic hybridization analysis of lung adenocarcinoma in sixty never smokers and identified fourteen new minimal common regions (MCR) of gain or loss, of which five contained a single gene (MOCS2, NSUN3, KHDRBS2, SNTG1 and ST18). One larger MCR of gain contained NSD1. One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FISH showed that the amplicon containing FUS was joined to the next telomeric amplicon at 16p11.2. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, CDKN2B, EGFR, ERBB2, MDM2, MDM4, MET, MYC and KRAS. Unsupervised hierarchical clustering with adjustment for false-discovery rate revealed clusters differing by the level and pattern of aberrations and displaying particular tumor characteristics. One cluster was strongly associated with gain of MYC. Another cluster was characterized by extensive losses containing tumor suppressor genes of which RB1 and WRN. Tumors in that cluster frequently harbored a central scar-like fibrosis. A third cluster was associated with gains on 7p and 7q, containing ETV1 and BRAF, and displayed the highest rate of EGFR mutations. SNP array analysis validated copy-number aberrations and revealed that RB1 and WRN were altered by recurrent copy-neutral loss of heterozygosity.

Conclusions

The present study has uncovered new aberrations containing cancer genes. The oncogene FUS is a candidate gene in the 16p region that is frequently gained in never smokers. Multiple genetic pathways defined by gains of MYC, deletions of RB1 and WRN or gains on 7p and 7q are involved in lung adenocarcinoma in never smokers.     

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.     

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.     

Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis

This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.Plant primary metabolism is a complex process where many interacting pathways must be finely coordinated and integrated in order to achieve proper plant development and acclimation to the environment. An example of such complexity is the biosynthesis of the amino acid l-Ser, which takes place in at least two different organelles and by different pathways. This amino acid is essential for the synthesis of proteins and other biomolecules needed for cell proliferation, including nucleotides and Ser-derived lipids, such as phosphatidylserine and sphingolipids. Additionally, d-Ser has been attributed a signaling function in male gametophyte-pistil communication (Michard et al., 2011).Despite the important role played by Ser in plants, the biological significance of the coexistence of several Ser biosynthetic pathways and how they interact to maintain amino acid homeostasis in cells is not yet understood. Three different Ser biosynthesis pathways have been described in plants (Kleczkowski and Givan, 1988; Ros et al., 2013; Fig. 1). One is the glycolate pathway, which takes place in mitochondria and is associated with photorespiration (Tolbert, 1980, 1997; Douce et al., 2001; Bauwe et al., 2010; Maurino and Peterhansel, 2010). In this pathway, two molecules of Gly are converted to one molecule of Ser in a reaction catalyzed by the Gly decarboxylase complex and Ser hydroxymethyltransferase (Fig. 1). Ser synthesis through the glycolate pathway is obtained in green tissues during daylight hours (Tolbert, 1980, 1985; Douce et al., 2001), suggesting that alternative Ser biosynthesis pathways may be required in the dark and/or in nonphotosynthetic organs. In this respect, Ser can be synthesized through two nonphotorespiratory pathways (Kleczkowski and Givan, 1988), the plastidial phosphorylated pathway (Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001) and the so-called glycerate pathway, which synthesizes Ser by the dephosphorylation of 3-phosphoglycerate (3-PGA; Kleczkowski and Givan, 1988; Fig. 1). This latter pathway includes the reverse sequence of the section of the oxidative photosynthetic carbon cycle linking 3-PGA to Ser (3-PGA-glycerate-hydroxypyruvate-Ser), these reactions being catalyzed by putative enzymes such as 3-PGA phosphatase, glycerate dehydrogenase, Ala-hydroxypyruvate aminotransferase, and Gly hydroxypyruvate aminotransferase. Although the existence of enzymatic activities of this pathway has been demonstrated (Kleczkowski and Givan, 1988), its functional significance is unknown and genes coding for the specific enzymes of the pathway have not been characterized to date.Open in a separate windowFigure 1.Schematic representation of Ser biosynthesis in plants. The enzymes participating in each Ser biosynthetic pathway are listed separately. Photorespiratory pathway (glycolate pathway): GDC, Gly decarboxylase; SHMT, Ser hydroxymethyltransferase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, Ala-hydroxypyruvate aminotransferase. Phosphorylated pathway: PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Abbreviations used for metabolites are as follows: 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; THF, tetrahydrofolate; 5,10-CH₂-THF, 5,10-methylene-tetrahydrofolate. This figure is adapted from Cascales-Miñana et al. (2013).The plastidial phosphorylated pathway of serine biosynthesis (PPSB; Fig. 1), which is conserved in mammals and plants, synthesizes Ser via 3-phosphoserine utilizing 3-PGA as a precursor (Kleczkowski and Givan, 1988). Evidence for the existence of this pathway in plants stems from the isolation and characterization of its enzyme activities (Handford and Davies, 1958; Slaughter and Davies, 1968; Larsson and Albertsson, 1979; Walton and Woolhouse, 1986). The PPSB involves three enzymes catalyzing sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase (PSP; Fig. 1). Genes coding for some isoforms of these enzymes have been cloned and biochemically characterized in Arabidopsis (Arabidopsis thaliana; Ho et al., 1998, 1999a, 1999b; Ho and Saito, 2001).In humans, the PPSB plays a crucial role in cell proliferation control and oncogenesis (Bachelor et al., 2011; Locasale et al., 2011; Pollari et al., 2011; Possemato et al., 2011). The functional significance of the PPSB in plants has recently been unraveled by providing evidence for the crucial role of PSP1, the last enzyme of the pathway in embryo, pollen, and root development (Cascales-Miñana et al., 2013). However, the PPSB still requires further characterization. In order to gain a complete understanding of the PPSB function in plants, precise molecular, metabolic, and genetic knowledge of all the enzymes and genes of the pathway is needed. In this work, we follow a gain- and loss-of-function approach in Arabidopsis to characterize a family of genes coding for putative isoforms of PGDH, the first enzyme of the PPSB. Here, we identify the essential gene of this family and provide evidence for its crucial function in embryo and pollen development.     

Reactive Oxygen Species Production and Antioxidant Enzyme Expression after Epstein–Barr Virus Lytic Cycle Induction in Raji Cell Line

In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48?h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24?h (p?相似文献   

Sterilization and protection of protein in combinations of Camellia sinensis green tea extract and gamma irradiation

Sterilization of milk protein without heating is of great interest. Gamma irradiation is a very powerful method to decontaminated casein. Gamma-irradiation of proteins in aqueous media at doses higher than 5kGy is known to induce their aggregation (without oxygen) or degradation (in presence of oxygen). Camellia sinensis green tea extract addition before irradiation of caseins cow milk proteins was examined. It was found that the presence of C. sinensis green tea extract during irradiation in the presence of oxygen conditions prevented the protein aggregation even at doses higher than 10kGy, probably by scavenging oxygen radicals produced by irradiation. The protective role of C. sinensis green tea extract allowing the gamma-irradiation treatment of caseins cow milk proteins in solution, was asserted by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography inverse phase (RP-HPLC). The total viable microorganisms content evaluated by Plate Count Agar (PCA) incubation for 12h at 37°C, showed that caseins protein preparations gamma-irradiated remained sterile at a dose 2kGy in absence of C. sinensis green tea extract and at a dose lower than 2kGy in the presence of C. sinensis green tea extract.