Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNgamma) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNgamma was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology.
cirrhosis only one showed intense staining. In HBV and HCV, 5/26 were positive and 0/9 in normal [p<0.001]. In rat tissues, TLR7 was found in all HCC tumour cells only while the background either normal, dysplastic or cirrhotic was negative. Study 2. Using confocal microscopy, TLR7 was found in the cytoplasm and the nucleus of both HepG2 and Huh7 and with stimulation of TLR7 agonist the cellular proliferation significantly increased compared to control p<0.05. Conclusion The data show that TLR7 is highly expressed in human HCC's, animal model of HCC and in cell lines. Importantly, the background cirrhotic liver does not express TLR7. Their stimulation is associated with marked increase in proliferation. These data suggest that TLR7 may be a future target of therapy in HCC.Competing interests None declared. Introduction Degradation of the extracellular matrix is fundamental to tumour development, invasion and metastasis. Several protease families have been implicated in the development of a broad range of tumour types, including oesophago-gastric (OG) adenocarcinoma. The aim of this study was to analyse expression levels of all core members of the cancer degradome in OG adenocarcinoma, and to investigate the relationship between expression levels and tumour/ patient variables associated with poor prognosis. Methods Comprehensive expression profiling of the protease families [matrix metalloproteinases (MMPs), members of the ADAM metalloproteinase-disintegrin family (ADAMs)], their inhibitors [tissue inhibitors of metalloproteinase (TIMPs)], and molecules involved in the c-Met signalling pathway, was performed using quantitative real-time reverse transcription PCR in a cohort of matched malignant and benign peri-tumoural OG tissue (n¼25 patients). Data were analysed with respect to clinico-pathological variables (tumour stage and grade, age, sex and pre-operative plasma C-reactive protein level). Results Gene expression of MMP1, 3, 7, 9, 10, 11, 12, 16 and 24 was upregulated by factors greater than fourfold in OG adenocarcinoma samples compared with matched benign tissue (p<0.01). Expression of ADAM8 and ADAM15 correlated significantly with tumour stage (p¼0.048 and p¼0.044), and ADAM12 expression correlated with tumour grade (p¼0.011). Conclusion This study represents the first comprehensive quantitative analysis of the expression of proteases and their inhibitors in human OG adenocarcinoma. These findings implicate elevated ADAM8, 12 and 15 mRNA expression as potential prognostic molecular markers.
PMO-093
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