GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

BACKGROUND: Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model. CONCLUSION/SIGNIFICANCE: The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53

Surface-enhanced Raman scattering and electrochemical characterization of metal electrode-electrolyte interfaces in nonaqueous systems

Many important and interesting processes, which are essential components of electrochemical technologies, occur at the electrode/electrolyte interface. Using spectroscopic and electrochemical methods, this research elucidates the interfacial structure in nonaqueous solvents existing at metal electrodes. The behavior of trace interfacial H₂O and Li⁺ and Br⁻ ions at Ag electrodes in the normal alcoholic solvents methanol, ethanol, propanol, butanol and pentanol is examined using surface-enhanced Raman scattering (SERS) and differential capacitance measurements. SERS spectra in the ν(O-H) region exhibit four bands from interfacial H₂O molecules which are a sensitive function of the nature of the solvent, the residual H₂O concentration, and the electrode potential. The results of this study indicate that in the interface, H₂O molecules cluster around Li⁺ and Br⁻ ions and are not homogeneously dispersed. Br⁻ specific adsorption and interfacial solvent behavior in the series of alcohol solvents are interpreted quantitatively through differential capacitance and SERS experiments. Br⁻ surface coverage is calculated using the "Hurwitz-Parsons" analysis of differential capacitance-potential data in these media. These results indicate that almost a monolayer of Br⁻ adsorb on Ag electrodes, but much less Br⁻ ions adsorbs on Au electrodes at positive potentials. The potential of zero charge (pzc) is also quantitatively estimated from these measurements. The technique of "emersion" of an electrochemical interface from electrolyte has opened the door to new possibilities for fundamental electrochemical studies. The relationship between in-situ and emersed electrochemical systems is probed through the study of the interfacial structure of dimethyl sulfoxide (DMSO) on Ag electrodes. The results of this study indicate that pronounced restructuring of the DMSO in the interface is found in the vicinity of the pzc. The solvent orientation is generally retained upon emersion, but a negative emersion-induced potential shift in the interface is observed due to the reorientation of DMSO molecules at gas/solution boundary phase. These data represent the first spectroscopic evidence for the existence of this emersion-induced potential shift

Development and Application of Hydrophilic Interaction Liquid Chromatographic Stationary Phases

As an alternative high performance liquid chromatography technology, hydrophilic interaction liquid chromatography (HILIC) plays an important role in the separation of highly polar and ionic compounds such as amino acids, carbohydrates, peptides and so on. The development of HILIC stationary phases, which are considered as the core of chromatographic technology, is of great importance for the improvement of chromatographic separation selectivity and efficiency, influencing the application and generalization of HILIC. To date, a great variety of commercial and academic HILIC materials with abundant functional groups have been presented. According to the functional groups of chromatographic materials, the development of silica-based HILIC stationary phases including unmodified silica, amino-based, cyano-based, diol-based, amide-based, poly (succinimide) -based, saccharides-based and zwitterion-based stationary phases are reviewed in the present article. Meanwhile, the typical applications of HILIC stationary phases in polar drugs separation, bioanalysis, proteomics, metabolomics, etc. are included. With increasing diversity of HILIC materials, the selection of appropriate HILIC stationary phase for the resolution of complex compounds becomes difficult. The development of systematic test samples and methods to access the separation selectivity is of great value for understanding the interaction mechanism of HILIC stationary phases. Thus, the investigation involving test samples to evaluate separation efficiencies and selectivities in HILIC is also reviewed

A novel zwitterionic HILIC stationary phase based on "thiol-ene" click chemistry between cysteine and vinyl silica

A novel zwitterionic stationary phase with high hydrophilicity was facilely synthesized based on the "thiol-ene" click reaction between cysteine and vinyl silica, which exhibited great potential in the separation of oligosaccharides, peptides and basic compounds, as well as in the enrichment of glycopeptides

Preparation of glutathione-functionalized zwitterionic silica material for efficient enrichment of sialylated N-glycopeptides

A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples

Practical method for the definition of chromatographic peak parameters in preparative liquid chromatography

A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm(3). The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography

Preparation of glyco-silica materials via thiol-ene click chemistry for adsorption and separation

Glyco-silica materials were successfully developed based on thiol-ene click chemistry between alkene-saccharides and mercapto-silica, which behaved well in HILIC separation and exhibited high affinity to specific proteins