Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

GPS-ARM: computational analysis of the APC/C recognition motif by predicting D-boxes and KEN-boxes

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org.     

EBV encoded miR-BHRF1-1 potentiates viral lytic replication by downregulating host p53 in nasopharyngeal carcinoma

miRNAs (microRNAs) are a class of non-coding small RNAs. The Epstein-Barr-virus (EBV) encoded miR-BHRF1-1 is barely expressed in most nasopharyngeal carcinoma (NPC) cells with EBV latent infection. Here, we used a strategy of overexpression and inhibition of miR-BHRF1-1 and showed that miR-BHRF1-1 is involved in TPA-induced accumulation of EBV lytic proteins and viral copies in late lytic cycle. The data further suggested that the miR-BHRF1-1-potentiated induction of EBV lytic replication was accompanied by inhibiting p53 expression. Our results demonstrated that the EBV original pathogen miR-BHRF1-1 is involved in the control of EBV late lytic replication by directly targeting the host p53 gene.     

Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer

Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P = 0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P = 0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G₂/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback loop. Further, inhibition of polo-like kinase 1 could enhance the sensitivity of non-small cell lung cancer cells to taxanes or irradiation. Thus, polo-like kinase 1 might be a prognostic marker and a chemo- or radiotherapeutic target for non-small cell lung cancer.     

Screening,cultivation, and biocatalytic performance of <Emphasis Type="Italic">Rhodococcus boritolerans</Emphasis> FW815 with strong 2,2-dimethylcyclopropanecarbonitrile hydratase activity

In this work, a mild, efficient bioconversion of 2,2-dimethylcyclopropanecarbonitrile (DMCPCN) to 2,2-dimethylcyclopropanecarboxamide (DMCPCA) in distilled water system was developed. The isolate FW815 was screened using the enrichment culture technique, displaying strong DMCPCN hydratase activity, and was identified as Rhodococcus boritolerans based on morphological, physiological, biochemical tests and 16S rRNA gene sequencing. Cultivation outcomes indicated that R. boritolerans FW815 was a neutrophile, with a growth optimum of 28–32°C; its DMCPCN hydratase belonged to the Fe-type family, and was most active at 38–42°C, pH 7.0, with maximal activity of 4.51 × 10⁴ U g⁻¹ DCW. R. boritolerans FW815 was found to be DMCPCA amidase-negative, eliminating the contamination of dimethylcyclopropanecarboxylic acid. Moreover, it displayed high activity and acceptable reusability in the non-buffered distilled water system, comparable to those in pH 7.0 phosphate buffer (50.0 mmol l⁻¹).     

The Arabidopsis ARCP protein, CSI1, which is required for microtubule stability, is necessary for root and anther development

Armadillo repeat-containing proteins (ARCPs) are conserved across eukaryotic kingdoms and function in various processes. Regulation of microtubule stability by ARCPs exists widely in mammals and algae, but little is known in plants. Here, we present the functional characterization of an Arabidopsis thaliana ARCP, which was previously identified as Cellulose synthase-interactive protein1 (CSI1), and prove its crucial role in anther and root development. CSI1 is highly expressed in floral tissues, and knockout mutants of CSI1 (three allelic lines) accordingly exhibit defective anther dehiscence, which can be partially rescued by mammalian microtubule-stabilizer MAP4, suggesting that CSI1 functions by stabilizing the microtubular cytoskeleton. CSI1 binds microtubules in vitro, and immunofluorescence and coimmunoprecipitation studies confirmed the physical interactions between CSI1 and microtubules in vivo. Analysis using oryzalin, a microtubule-disrupting drug, further revealed the destabilized microtubules under CSI1 deficiency and confirmed the crucial role of CSI1 in microtubule stability. The dynamic change of CSI1 in response to dehydration strongly suggests the important function of CSI1 in dehydration-induced microtubule depolymerization and reorganization, which is crucial for anther development. These results indicate the pivotal role of CSI1 in anther development by regulating microtubule stability and hence cell morphogenesis.     

Identification of genes preferentially expressed in goat hair follicle anagen-catagen transition using suppression subtractive hybridization

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes in goat (Capra hircus) hair follicle anagen-catagen transition. The cDNA fragments, derived from SSH positive subtractive library (tester: anagen-catagen transition, driver: later anagen), were cloned into pEGM-T vector. Two hundred cDNA fragments screened from this library were subjected to identify forty-five unregulated isolates. Sequence analysis revealed that these fragments represented twenty-three genes. Blasting analysis with database in GenBank showed that twenty genes were previously clearly annotated, two were homologous to un-annotated expressed sequence tag (ESTs), and one might be novel. To identify characters of gene expression, seven genes in later anagen and anagen-catagen transition skin tissues were chosen for quantitative real-time PCR. Results indicated that expression of these seven genes varied much, reaching threefold among them, furthering indicating that expression of those genes was up-regulation in the anagen-catagen transition. We characterized expression levels of this potential novel gene and the goat ectodysplasin A during differential stages of hair cycle. These profiles suggested that these two genes might play a role in the goat secondary hair follicle cycle.