[DeSUMOylation of protein kinase B1 inhibits cell proliferation and metastasis of hepatocellular carcinoma].

Objective: To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells. Methods: The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining. Results: Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) µm and 89.44±8.36, (56.56±5.37) µm and 93.84±8.79, as well as (34.57±6.61) µm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05). Conclusions: Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.

Molecular characterization of classical and nonclassical MHC class I genes from the golden pheasant (Chrysolophus pictus).

Classical major histocompatibility complex (MHC) class I allelic polymorphism is essential for competent antigen presentation. To improve the genotyping efforts in the golden pheasant, it is necessary to differentiate more accurately between classical and nonclassical class I molecules. In our study, all MHC class I genes were isolated from one golden pheasant based on two overlapping PCR amplifications. In total, six full-length class I nucleotide sequences (A-F) were identified, and four were novel. Two (A and C) belonged to the IA1 gene, two (B and D) were alleles derived from the IA2 gene through transgene amplification, and two (E and F) comprised a third novel locus, IA3 that was excluded from the core region of the golden pheasant MHC-B. IA1 and IA2 exhibited the broad expression profiles characteristic of classical loci, while IA3 showed no expression in multiple tissues and was therefore defined as a nonclassical gene. Phylogenetic analysis indicated that the three IA genes in the golden pheasant share a much closer evolutionary relationship than the corresponding sequences in other galliform species. This observation was consistent with high sequence similarity among them, which likely arises from the homogenizing effect of recombination. Our careful distinction between the classical and nonclassical MHC class I genes in the golden pheasant lays the foundation for developing locus-specific genotyping and establishing a good molecular marker system of classical MHC I loci.

Major histocompatibility complex class II polymorphisms in forest musk deer (Moschus berezovskii) and their probable association with purulent disease.

Genes of the major histocompatibility complex (MHC) family are crucial in immune responses because they present pathogenic peptides to T cells. In this study, we analysed the genetic variation in forest musk deer (Moschus berezovskii) MHC II genes and its potential association with musk deer purulent disease. In total, 53 purulent disease-susceptible and 46 purulent disease-resistant individuals were selected for MHC II exon 2 fragment analysis. Among them, 16 DQ alleles and four additional DR alleles were identified, with DQ exon 2 fragments displaying a low level of polymorphism. The nonsynonymous substitutions exceeded the synonymous substitutions in the peptide-binding sites of DQA2, DQB1 and DQB2. Then, 28 MHC II alleles were used to analyse the distribution patterns of purulent disease between the susceptible and resistant groups. Among them, three alleles (DQA1*01, DQA1*02 and DQA2*04) were found to be resistant, and five alleles (DRB3*07, DQA1*03, DQA1*04, DQA2*05 and DQA2*06) were found to increase susceptibility. Additionally, three haplotypes were found to be putatively associated with musk deer purulent disease. However, these three haplotypes were only found in the resistant or susceptible group, and their frequencies were low. The results from our study support a contributory role of MHC II polymorphisms in the development of purulent disease in forest musk deer.

Effect of monoclonal antibody on expression of lipid metabolism related genes in porcine adipocytes.

In order to study the mechanism of monoclonal antibody (McAb) against a porcine 40-kDa adipocyte-specific plasma membrane protein in reducing fat deposition, porcine primary adipocytes were treated with the McAb during the process of adipocyte differentiation; its effect on expression of lipid metabolism related genes was investigated. Adipocytes were treated with 1-methyl-3-isobutylmethylxanthine (IDX) plus 10 microg/mL of the McAb or without McAb. The mRNA levels of adipocyte differentiation related genes (PPARgamma and C/EBPalpha), lipid metabolism related genes (FAS, HSL, CPT-1B, DGAT and A-FABP) and adiponectin gene (AdipoQ) were determined using real-time quantitative PCR. The results showed that the differentiated adipocyte number and triglyceride (TG) content in adipocytes treated with the McAb were lower than that in cells without McAb during the whole process of adipocyte differentiation. The McAb significantly reduced mRNA expression of PPARgamma, C/EBPalpha, FAS, DGAT, A-FABP and adiponectin genes, but increased mRNA expression of HSL and CPT-1B genes during the medium and latter stage of adipocyte differentiation. This suggested that the McAb decreased triglycerol accumulation in adipocyte by both inhibiting adipocyte differentiation and regulating lipid metabolism, especially at the medium and latter stage of porcine adipocyte differentiation.

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization.

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.

Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection.

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin-antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody-tracer complex during the separation. The calibration curves covered a concentration range 0.05-1.0 microg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.

Fluorescence polarization studies of affinity interactions in capillary electrophoresis.

Capillary electrophoresis (CE) combined with molecular recognition for ultrasensitive bioanalytical applications often requires the formation of stable complexes between an analyte and its binding partner. Previous studies of binding interactions using CE involve multiple-step titration experiments and are time-consuming. We describe a simple method based on laser-induced fluorescence polarization (LIFP) detection for CE separation, which allows for on-line monitoring of affinity complex formation. Because fluorescence polarization is sensitive to changes in the rotational diffusion arising from molecular association or dissociation, it is capable of providing information on the formation of affinity complexes prior to or during CE separation. Applications of the CE/LIFP method to three binding systems including vancomycin and its antibody, staphylococcal enterotoxin A and its antibody, and trp operator and trp repressor were demonstrated, representing peptide-protein, protein-protein, and DNA-protein interactions. The affinity complexes were readily distinguished from the unbound molecules on the basis of their fluorescence polarization. The relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs.

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays.

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE-LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone.

Effect of electrical double-layer overlap on the electroosmotic flow in packed-capillary columns.

A plug electroosmotic velocity profile is generally assumed to be characteristic of capillary electrochromatography. However, this ideal plug flow may be illusive in some experiments with packed-capillary columns due to overlap of electrical double layers in flow channels. We report here a theoretical analysis of the double-layer overlap effects in packed-capillary columns, which is based on Rice and Whitehead's theory of electroosmotic flow combined with a capillary tube model for porous packing. The results show that the electroosmotic velocity under the influence of double-layer overlap depends strongly on the operating parameters, which increases with the column porosity, the particle diameter, and the electrolyte concentration.

Retention behavior of ionizable isomers in reversed-phase liquid chromatography: a comparative study of porous graphitic carbon and octadecyl bonded silica.

The retention behaviors of 36 positional isomers of ionizable substituted benzene compounds have been compared on two different packing materials: porous graphitic carbon (PGC) and octadecyl bonded silica (ODS) using 35% aqueous acetonitrile as the mobile phase. The effect of the mobile phase pH on the solute retention was studied over a range of pH values from pH 2.0 to 7.0. The retention as a function of pH was modeled using equations based on solute ionization. With PGC, the theoretical equations fitted the observed retention data for each class of solute, indicating that the retention mechanism was uniform over the whole pH range. However, with ODS, only the acidic solutes showed agreement with the theoretical model; for the amine-containing compounds, serious deviations from the theory were observed, suggesting that strongly acidic silanols gave added retention at low mobile phase pH. Overall, PGC demonstrated a higher selectivity toward positional isomers than ODS. This was attributed to the greater steric discriminating ability arising from the flat surface of the PGC compared with the more fluid nature of the ODS bonded phase.