Establishment of a Model of Microencapsulated SGC7901 Human Gastric Carcinoma Cells Cocultured with Tumor-Associated Macrophages.

The important factors of poor survival of gastric cancer (GC) are relapse and metastasis. For further elucidation of the mechanism, a culture system mimicking the microenvironment of the tumor in humans was needed. We established a model of microencapsulated SGC7901 human GC cells and evaluated the effects of coculturing spheres with tumor-associated macrophages (TAMs). SGC7901 cells were encapsulated in alginate-polylysine-sodium alginate (APA) microcapsules using an electrostatic droplet generator. MTT assays showed that the numbers of microencapsulated cells were the highest after culturing for 14 days. Metabolic curves showed consumption of glucose and production of lactic acid by day 20. Immunocytochemistry confirmed that Proliferating Cell Nuclear Antigen (PCNA) and Vascular Endothelial Growth Factor (VEGF) were expressed in microencapsulated SGC7901 cells on days 7 and 14. The expression of PCNA was observed outside spheroids; however, VEGF was found in the entire spheroids. PCNA and VEGF were increased after being cocultured with TAMs. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions were detected in the supernatant of microencapsulated cells cocultured with TAMs but not in microencapsulated cells. Our study confirms the successful establishment of the microencapsulated GC cells. TAMs can promote PCNA, VEGF, MMP-2, and MMP-9 expressions of the GC cells.

[Detection and genotyping of rotavirus in diarrhea infant feces in Chengdu].

OBJECTIVE: To study the genotype of rotavirus and the genetic variations of the major neutralization antigen VP4 of group A rotavirus in fecal samples from infants with diarrhea in Chengdu, Sichuan province, China. METHODS: The fecal specimens were collected from infant patients with diarrhea in the spring of 2010 at West China Second University Hospital. Reverse transcription polymerase chain reaction (RT-PCR) was performed to identify rotavirus G serotypes and P genotypes. VP4 gene fragments of the virus were amplified from two strains drawn randomly from the prevailing genotype and cloned into a T-A clone vector to generate the recombinants for sequencing. RESULTS: A group rotaviruses were detected in 13 of 75 specimens (17.3%). Serotype G1 was the predominant type (7/13) and two were serotype G3, four strains' serotypes were unidentified. Analysis of P gene demonstrated that genotype P [8] was the predominant type (6/13), whereas only two P[4] genotype were detected and genotypes for two strains were not determined. G1P [8] was the predominant type of G/P dominance combination (5/13). Sequencing results of the VP4 gene for the analyzed two strains implied that they were genotype P[8] with a 97% homology in sequence. Compared with the standard strain, homologies were also more than 90%. CONCLUSION: Rotavirus is one of the major etiological agents of viral diarrhea among infants in Chengdu. G1 was the dominant type G in Chengdu. G1P[8] was the predominant type of G/P dominance combination.

Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells.

Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis.

Efficient sequence-directed psoralen targeting using pseudocomplementary Peptide nucleic acids.

A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.

[Determination of cyclovirobuxine-D in huangyangning tablets by RP-HPLC with gradient elution].

OBJECTIVE: To establish a new method for the determination of cyclovirobuxine-D in huangyangning tablets. METHOD: Agilent Zorbax Extend C18 (4.6 mm x 250 mm, 5 microm) column with temperature 40 degrees C was used. The mobile phase was 0.3% diethylamine methanol solution and 0.3% diethylamine water solution, with a linear gradient of 0.3% diethylamine methanol solution from 78% to 95% in 9 minutes, and then maintained for 6 minutes. The flow rate was 0.8 mL x min(-1). Evaporative light scattering detector was used. RESULT: The calibration curve was linear at a range of 0.57-11.44 microg for the cyclovirobuxine D (r = 0.9996). The average recovery was 97.2% and RSD was 1.1% (n = 9). CONCLUSION: This method is rapid, simple, and reproducible, and can be used as a quality control method for this preparation.

[Prokaryotic expression and purification of recombination Salmonella invasion protein].

OBJECTIVE: To express recombinant Salmonella invasion protein A(InvA) in E. coli and purify it. METHODS: The invA gene of Salmonella was amplified by PCR from the Salmonella genome and cloned into expression vector pET-30c (+) to generate the pET-invA recombinants. They were comfirmed by restriction endonuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). After inducing with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was purified via Ni-NTA affinity chromatography under denature conditions. The recombinant proteins were analyzed with SDS-PAGE and Western blot. RESULTS: The pET-invA recombinant for Salmonella was successfully constructed and the recombinant InvA protein was expressed in E. coli at a relatively high level. The SDS-PAGE results for the purified recombinant protein demonstrated that the purified protein had reached the electrophoresis purity. CONCLUSION: The successful expression of the Salmonella InvA protein will be very helpful for the further study on its antigenicity, immunological activity, and the development of rapid detection methods for Salmonella strains.

[Primarily study on a multiplex PCR-based system for the rapid detection of Salmonella spp., Shigella spp. and Escherichia coli O157:H7].

OBJECTIVE: To establish a multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and Escherichia coli 0157:H7 in 12 hours. METHODS: After 6 h nonselective enrichment in BPW, DNA template were prepared at 100 degrees C for 10 min. Three sets of primers were designed to amplify the gene segments of invA of Salmonella spp, ipaH of Shigella spp, and uidA of E. coli 0157:H7, and the products were analyzed by electrophoresis. At the same time, this system was optimized, and the specificity and sensitivity of this system were evaluated. RESULTS: Three target bacteria were detected in 12 h by using this multiplex PCR-based system. The sensitivity of it was up to 10-30 cfu/ml, and the high specificity was demonstrated by detecting 23 target stains and 15 non-target stains. CONCLUSION: A rapid, specific, and sensitive multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and E. coli 0157:H7 in 12 h has been studied primarily.

Proteasomal degradation of human peptidyl prolyl isomerase pin1-pointing phospho Bcl2 toward dephosphorylation.

Microtubule inhibitor-induced Bcl2 phosphorylation is detrimental to its antiapoptotic function. Phosphorylation of Bcl2 predominantly occurs on two serine residues (70 and 87) in cells arrested at G2-M phase by microtubule disarraying agents. Phospho Bcl2 can associate with a cis-trans peptidyl prolyl isomerase, Pin1. Pin1 and its homologues are known to target the proline residue carboxyl terminal to the phosphorylated threonine or serine residue of mitotic phosphoproteins, such as Bcl2. However, it was not clear how an extranuclear protein could associate with nuclear Pin1. The confocal images of the immunofluorescence studies employing phospho Bcl2-specific antibody developed in the laboratory demonstrated the translocation of phospho Bcl2 inside the nucleus. Interestingly, proteasomal degradation of Pin1 facilitates dephosphorylation of phospho Bcl2 due to longer exposure of Taxol. Here we show for the first time that proteasomal degradation of Pin1 is the key factor to determine the fate of phosphoforms of Bcl2. When Pin1 is degraded by proteasomes, phospho Bcl2 is converted to its native form. Thus, transient conformational change of Bcl2 due to association with peptidyl prolyl isomerase can contribute to irreversible apoptotic signaling.