Abstract:Lipidomics, a newly emerging field, is systems-level analysis and characterization of lipids and their interacting moieties. Lipids play essential roles in several cellular processes, such as membrane transport, sorting and cell signaling. Previous studies have revealed possible links between deregulated lipid metabolism and a wide variety of diseases, including diabetes, obesity, cancer, as well as neurodegenerative diseases. All these make the field of lipidomics a promising area of biological research. In fact, lipidomic research has been applied to many fields, such as drug development, molecular biology, molecular pathology, functional genomics, nutriology, environmental science, and so on. The progress in the field of lipidomics, including the present situation and the future application, was summarized.

Abstract:Inflammasome is a multiprotein complex and oligomeric platform that dimerizes and thereby activates cysteinyl aspartate-specific protease (caspase)-1. Caspase-1, also known as the interleukin (IL)-1β-converting enzyme (ICE), is the prototypical member of the inflammatory caspases and involved in regulating maturation of IL-1β, IL-18, and IL-33, key cytokines for the recruitment and engagement of inflammatory cells. Nucleotide-binding and oligomerization domain-like receptors (NLRs), which detect intracellular pathogens or other ‘alarms’, promote the assembly of inflammasome. Some evolutionary aspects and biochemical studies were reviewed, underlining the role of inflammasomes in infection as well as inflammatory diseases.

Abstract:Endothelial progenitor cells have great prospect for treatment of many diseases. However, derivation of endothelial progenitor cells in vitro is a major restriction for the clinical treatment. Human embryonic stem cells (hESCs) may become an alternative source of endothelial progenitor cells because of their high proliferation capability and in vitro pluripotency. Although, there are many challenges for differentiating hESCs to endothelial progenitor cells in vitro. Current understanding of this subject from recent discoveries in this field was summarized. Future work will be needed to translate these in vitro findings to clinical applications.

Abstract:Folic acid deficiency induces congenital malformations. Dihydrofolate reductase (DHFR) plays key roles in folic acid metabolism. The dysfunction of DHFR results in the inhibiting of folic acid. In vertebrate, pharyngeal arches are the progenitor of craniofacial structure and cardiac out flow tract. By using zebrafish as the animal model and coupling with gene knock-down and over-expression technologies, the role of dihydrofolate reductase gene (DHFR) in the development of pharyngeal arches were explored. In DHFR knock-down embryos, the malformations of pharyngeal arches and palates were showed by paraffin section and alcian blue staining. DHFR over-expressing can rescue these malformations. TBX1 and HAND2 are two key transcription factors in the development of pharyngeal arches. Whole-mount in situ hybridization and Real-time PCR were performed to detect the expression levels of TBX1 and HAND2. The expressions of TBX1 and HAND2 were reduced in DHFR knock-down group. DHFR over-expression can increase expressions of TBX1 and HAND2. These results demonstrated that DHFR was essential for the development of pharyngeal arches and DHFR regulated the development of pharyngeal arches by effecting the expressions of TBX1 and HAND2.

Abstract:To investigate the effect of Aβ on mitochondrial dysfunction, cells stably transfected by human amyloid precursor protein 695 (APP) and cells stably co-transfected by familial Alzheimer’s disease (FAD)-linked Swedish mutant of APP695 gene plus ΔE9 deleted presenilin1 gene (swe. Δ9) were used to study the mitochondrial function. Swe. Δ9 cells showed more reduce in mitochondrial membrane potential, altered complexⅣ activity, mitochondrial membrane fluidity and ATP content. However, these parameters in APP cells were lower than those in N2a cells stably transfected with empty vector. The similar results were observed in transgenic mice. APP/PS1M146V/+ mice showed more reduced complexⅣ activity and ATP content than Tg2576 mice. These data indicated that the dose-dependent effect of Aβ on mitochondrial dysfunction.

Abstract:Alternative splicing of tau exon 10 generates tau isoforms with 3 or 4 microtubule-binding repeats. In normal adult human brain, approximately equal levels of 3R-tau and 4R-tau are expressed, which is required for maintaining normal brain functions. 9G8, one member of SR protein family, is involved in the splicing of many genes. The function of 9G8 is highly regulated by the phosphorylation. It was reported that GSK-3β regulates the alternative splicing of tau exon 10. Mini-tau gene were used to study the regulation of 9G8 on tau exon 10 splicing and the effect of GSK-3β on 9G8-mediated tau exon 10 splicing. It was found that 9G8 inhibited the inclusion of tau exon 10. GSK-3β phosphorylated 9G8 in vitro and interacted with 9G8. Overexpression of GSK-3β inhibited 9G8-mediated tau exon 10 inclusion.

Abstract:To investigate the inhibitory function of NPCEDRG, a novel tumor suppressor gene, in nasopharyngeal carcinoma (NPC), Tet-on system was introduced. Two plasmids were co-transfected into CNE2 cells, then a transgene CNE2 cell line, which had rapidly inducible and reversible expression of NPCEDRG, was obtained after two selections with G418 or hygromycin. NPCEDRG mRNA expression was detected via RT-PCR assay. Dox was used to induce the expression of NPCEDRG and a cell clone sensitive to Dox was selected. The best-induced concentration was determined with different concentration of Dox induction. Cell morphology observation, growth curves, clone formation rate and cell cycle distribution were detected after NPCEDRG restoration expression with Dox induction. After the restoration of NPCEDRG expression, the degree of CNE2 differentiation was higher than ever, the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed, and the cell percentage in G0/G1 phase increased, while percentage of cells entering the S and G2 phase decreased. This data indicates that an abnormality of NPCEDRG expression is associated with nasopharyngeal carcinogenesis and that it may play an important role in inducing cell differentiation, controlling cell growth and regulating the cell cycle.

Abstract:To study the effect of α1,2-fucosyltransferase gene transfection on p38MAPK signaling pathway- mediated apoptosis in ovarian carcinoma RMG-I cells. The localization of p38MAPK and p-p38MAPK was detected by immunofluorescence in RMG-I and RMG-I-H cells. The expression of p38MAPK and p-p38MAPK was analyzed by RT-PCR and Western blot, respectively. For inhibition assay, anti-Lewis y antibody was used to assess the change of p38MAPK of mRNA level in RMG-I-H cells by RT-PCR and Western blot. Using 0.1%DMSO as control, the apoptosis rate was detected by flow cytometry(FCM) in SB203580 treated RMG-I-H cells. Simultaneously, the expression of p38MAPK and caspase-3 was analyzed by RT-PCR and Western blot. Further more, the expression of p38MAPK and caspase-3 by RT-PCR after Carboplatin and/or SB203580 treatment were studied. Results showed that immunofluorescence staining of p38MAPK and p-p38MAPK in RMG-I and RMG-I-H cells showed cytoplasmic localization and nuclear localization, respectively, and the level of p-p38MAPK mRNA in RMG-I-H cells is significantly higher than that in RMG-I cells(P < 0.05), while the expression of p-p38MAPK mRNA decreased after anti-Lewis y antibody treatment (P < 0.05). FCM showed that the apoptosis rate increased in SB203580 treated RMG-I-H cells(P < 0.05). The mRNA level of p38MAPK and caspase-3 increased by treatment with Carboplatin. The mRNA level of caspase-3 also elevated by treatment with SB203580. In conclusion, high expression of Lewis y inhibits apoptosis in ovarian cancer cells, probably due to involvement of Lewis y in regulating p38MAPK signaling pathway, thereby causing drug resistance.

Abstract:Chk1 and Chk2 play major role in cell cycle checkpoint signaling pathway, which are mainly involved in G2/M cell cycle checkpoint signal transduction. Firstly, the siRNA targeting at Chk1 or Chk2 gene was transfected into human gastric cancer BGC823 cells for 24 h before 15 mg/L DADS was added. Then, the mRNA and protein expression of Chk1 or Chk2 was detected by Real-time PCR and Western-blot respectively. Cell cycle rates and expressions of CDC25C and cyclinB1 were determined by FCM and Western blot respectively. The results showed that the Chk1 or Chk2 expression was inhibited in Chk1 or Chk2 siRNA-transfected group, in which the Chk1 or Chk2 expression at mRNA level was reduced 84.7% and 69.0% respectively and the protein expression of Chk1 or Chk2 was reduced 73.4% and 78.5% respectively as compared with that in empty control group (P < 0.05). Then it was investigated whether Chk1 and Chk2 proteins could influence cell cycle regulation by knocking down their expressions in BGC823 cells. Further investigation revealed that inhibition of the Chk1 expression in Chk1 siRNA transfected group cut down the proportion of the cells in G2/M phase from 58.1% to 10.4% in BGC823 cells after 24 h induced by DADS (P < 0.05).While inhibition of the Chk2 expression in Chk2 siRNA transfected group had little effect on G2/M arrest after treatment with DADS(P > 0.05). Western blot showed that although DADS decreased expression of CDC25C and cyclinB1 in untransfected cells, inhibition of expression of CDC25C and cyclinB1 treated by DADS was blocked by Chk1 gene silence (P < 0.05). On the contrary, Chk2 gene silence can not do so. These results suggest Chk1 gene silence could abrogate G2/M arrest induced by DADS in BGC823 cell line, and Chk1/CDC25C/cyclinB1 pathway was involved at the G2/M arrest induced by DADS.

Abstract:ATP-binding Cassette Transporter A1 (ABCA1) plays a critical role in the reverse cholesterol transport (RCT). Previous studies showed that paraoxon, the active metabolite of organophosphorus insecticide, increased cholesterol retention in macrophages. However, its underlying mechanisms remain to be elucidated. The effect of paraoxon on ABCA1 expression and ABCA1-dependent cholesterol efflux was investigated, and then the role of cyclic adenosine monophospate (cAMP) signaling pathway in the regulation of ABCA1 expression and ABCA1-mediated cholesterol efflux was examined by paraoxon in RAW 264.7 macrophage-derived foam cells. Results showed that paraoxon significantly down regulated ABCA1 expression and reduced ABCA1-dependent cholesterol efflux and increased the levels of the total, free and esterified cholesterols in a time- and dose-dependent manner. Paraoxon also markedly reduced cAMP level and decreased adenylate cyclase (AC) activity and increased cAMP-specific phosphodiesterase (PDE) activity. Furthermore, cAMP analogs dibutyryl cyclic adenosine monophosphate (dBcAMP) markedly compensated the down-regulation of ABCA1 expression and partly compensated the reduction of ABCA1-mediated cholesterol efflux induced by paraoxon. Also, both adenylate cyclase agonist forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) markedly compensated the suppression effect on cAMP level induced by paraoxon. In conclusion, the results mentioned above suggest that paraoxon down regulates ABCA1 expression and decreases ABCA1-mediated cholesterol efflux through cyclic AMP signaling pathway in RAW 264.7 macrophage-derived foam cells.

Abstract:To obtain a high efficiency Pichia pastoris cell surface display system, two new systems based on two different anchor proteins derived from Saccharomyces cerevisiae Flo1 protein (Flo1p) were constructed respectively. The N-terminal anchor system could make the foreign lipase displayed on the P. pastoris cell surface with its C terminus free by fusion with an anchor protein containing N-terminal flocculation functional domain of Flo1p (874 residues, FS) , which was able to adhere to the cell surface via noncovalent interaction with the mannan chain of the cell wall. Conversely, the foreign lipase can kept its N terminus free in another C-terminal anchor system, which utilizes a GPI-attachment signal domain located at C-terminal region of Flo1p (1 101 residues, FL) as anchor protein. Using these systems above, recombinant R. miehei lipase with a pro region (ProRML) , which had its active site near the C-terminus, was displayed on the P. pastoris cell surface, and two surface-displayed RML, named as FSR and FLR, were obtained. Cell-surface display of the RML via Fs or FL anchor system was confirmed by flow cytometer and laser scanning confocal microscope. A strong fluorescence was clearly observed in recombinant yeast cells harboring pKFSR( pKFS-RML), but no fluorescence was detected in the yeast cells harboring pKFLR(pKFL-RML) . The hydrolytic activity of FSR reached 105. 3 U/g·[dry cell weight] with p-Nitrophenyl caprylate (pNPC) as the substrate, which is 2 times as high as that of FLR. In addition, the cell-surface display systems based on FS or FL endowed the displayed RML with different enzymatic properties. The surface-displayed RML with its C-terminus free (FSR) showed a better catalytic performance at temperature, pH and thermostability than the surface-displayed RML with its N-terminus free (FLR) did. The results suggest that the surface display of RML based on FS anchor system is more promising and more effective, especially for N-terminal immobilization of target enzyme whose catalytic site is near the C terminus.

Abstract:Traditional analysis of gene expression data focused on identifying differentially expressed and co-expressed genes, which didn′t take known interactions into consideration. In recent years, many methods have been developed to identify active subnetwork by integrating protein interaction networks with gene expression profiles. Current approaches failed to take full account of both difference and correlation in gene expression that may lead to false positive results. A new algorithm is proposed for identifying active subnetwork by considering both difference and correlation of gene expression profile. The algorithm is employed in the process of gene expression profiles of human immunodeficiency virus infection. The results showed that the algorithm can identify the active subnetwork that has extremely high biologically functional connectivity with human immunodeficiency virus, and effectively avoid the bias introduced by considering differences of gene expression profiles only, i.e., genes less differentially expressed are also included due to high correlations in gene expression.

Abstract:A method that could separate the subclasses of high density lipoprotein (HDL) rapidly by microfluicic chip electrophoresis was reported. Combined with laser induced fluorescence detection system, using the microfluicic chip designed independent, using 40 mmol/L Tricine, 50 mmol/L MEG and 0.2 mmol/L SDS (pH 8.5) as sample buffer solutions, using 40 mmol/L Tricine, 50 mmol/L MEG and 0.01 mmol/L SDS (pH 8.5) as separation buffer solutions, the two subclasses, HDL₃ and HDL₂ could be separated in 4 min. HDL subclasses could be separated with high efficiency and good reproducibility. The operation was easy and cost little. This method could meet the demand of the clinical examination of HDL subclasses, with good clinical value.

Abstract:Protein quantitative research has become a hot topic in proteomics and a main approach to discover biological protein functions. The technique based on isotope labeling and biological mass spectrometry analysis is one of the most commonly used strategies for protein quantification. In recent years, with the development of mass spectrometry techniques, inductively coupled plasma mass spectrometry (ICP-MS) becomes a powerful tool for research and measurement of elements, and may be enabling new insights and broad application in protein quantification. The advance of ICP-MS based techniques in the research of protein quantification was reviewed.