NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91^phox was dose-dependent. Meanwhile, the cytoplasmic subunit p47^phox was translocated to the cell membrane and localized with p22^phox and gp91^phox to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.     

Herpes Virus MicroRNA Expression and Significance in Serous Ovarian Cancer

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Inhibition of serine proteases by functionalized sulfonamides coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

A challenge associated with drug design is the development of selective inhibitors of proteases (serine or cysteine) that exhibit the same primary substrate specificity, that is, show a preference for the same P(1) residue. While these proteases have similar active sites, nevertheless there are subtle differences in their S and S' subsites which can be exploited. We describe herein for the first time the use of functionalized sulfonamides as a design and diversity element which, when coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold yields potent, time-dependent inhibitors of the serine proteases human leukocyte elastase (HLE), proteinase 3 (PR 3) and cathepsin G(Cat G). Our preliminary findings suggest that (a) appending to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold recognition and diversity elements that interact with both the S and S' subsites of a target protease may result in optimal enzyme selectivity and potency and, (b) functionalized sulfonamides constitute a powerful design and diversity element with low intrinsic chemical reactivity and potentially wide applicability.     

Endoplasmic Reticulum Stress in Heat- and Shake-Induced Injury in the Rat Small Intestine

We investigated the mechanisms underlying damage to rat small intestine in heat- and shake-induced stress. Eighteen Sprague-Dawley rats were randomly divided into a control group and a 3-day stressed group treated 2 h daily for 3 days on a rotary platform at 35°C and 60 r/min. Hematoxylin and eosin-stained paraffin sections of the jejunum following stress revealed shedding of the villus tip epithelial cells and lamina propria exposure. Apoptosis increased at the villus tip and extended to the basement membrane. Photomicrographs revealed that the microvilli were shorter and sparser; the nuclear envelope invaginated and gaps in the karyolemma increased; and the endoplasmic reticulum (ER) swelled significantly. Gene microarray analysis assessed 93 differentially expressed genes associated with apoptosis, ER stress, and autophagy. Relevant genes were compiled from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Forty-one genes were involved in the regulation of apoptosis, fifteen were related to autophagy, and eleven responded to ER stress. According to KEGG, the apoptosis pathways, mitogen-activated protein kinase(MAPK) signaling pathway, the mammalian target of rapamycin (mTOR) signaling pathway, and regulation of autophagy were involved. Caspase3 (Casp3), caspase12 (Casp12), and microtubule-associate proteins 1 light chain 3(LC3) increased significantly at the villus tip while mTOR decreased; phosphorylated-AKT (P-AKT) decreased. ER stress was involved and induced autophagy and apoptosis in rat intestinal damage following heat and shake stress. Bioinformatic analysis will help determine the underlying mechanisms in stress-induced damage in the small intestine.     

Molecular mapping of stripe rust resistance gene YrCH42 in Chinese wheat cultivar Chuanmai 42 and its allelism with Yr24 and Yr26

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat (Triticum aestivum L.) worldwide. The objectives of this study were to map a stripe rust resistance gene in Chinese wheat cultivar Chuanmai 42 using molecular markers and to investigate its allelism with Yr24 and Yr26. A total of 787 F₂ plants and 186 F₃ lines derived from a cross between resistant cultivar Chuanmai 42 and susceptible line Taichung 29 were used for resistance gene tagging. Also 197 F₂ plants from the cross Chuanmai 42×Yr24/3*Avocet S and 726 F₂ plants from Chuanmai 42×Yr26/3*Avocet S were employed for allelic test of the resistance genes. In all, 819 pairs of wheat SSR primers were used to test the two parents, as well as resistant and susceptible bulks. Subsequently, nine polymorphic markers were employed for genotyping the F₂ and F₃ populations. Results indicated that the stripe rust resistance in Chuanmai 42 was conferred by a single dominant gene, temporarily designated YrCH42, located close to the centromere of chromosome 1B and flanked by nine SSR markers Xwmc626, Xgwm273, Xgwm11, Xgwm18, Xbarc137, Xbarc187, Xgwm498, Xbarc240 and Xwmc216. The resistance gene was closely linked to Xgwm498 and Xbarc187 with genetic distances of 1.6 and 2.3 cM, respectively. The seedling tests with 26 PST isolates and allelic tests indicated that YrCH42, Yr24 and Yr26 are likely to be the same gene.G.Q. Li and Z.F. Li contributed equally to the work.