Abstract:Virus infection or interferon can stimulate robust expression of the protein ISG15 that is encoded by interferon stimulated gene 15, which was the first unbiquitin-like molecule identified two decades ago. While ubiquitin and its many important functions have been well established, the functions of ISG15 and its post-translational conjugation are still largely unknown . Recently, some specific enzymes have been identified to be involved in the ISG15 modification system, suggests that ISG15 and its modification system play important roles in the innate immune response and regulation of interferon signaling. The history of ISG15 discovery and its biochemical characterization were briefly introduced. Then such topics as the ISG15 gene expression and the ISG15 modification will be focued on, and finally summarize new findings which have implications for ISG15 and its modification system in immunology and interferon signal transduction were summarized.

Abstract:Genetical genomics is combining of microarry technology and quantitative trait loci (QTL) analysis, mapping expression QTL (eQTL) in global level. It provides a novel procedure for revealing the molecular mechanism and regulatory network of complex trait. The conception and strategy of genetical genomics were brought forward by Janson and Nap in 2001. By now, genetical genomics has been applied on yeast, mouse, human and plant such as maize. These results indicated that the variation of expression level of genes is heritable complex trait. Expression QTLs are classified into cis-acting and trans-acting. Cis-acting eQTL is located on the same genomic region of the regulated gene, meaning the variation of mRNA level could be due to the polymorphism in the gene itself. Trans-acting eQTL is located on other genomic region, meaning other gene controls the variation of mRNA level. The integration of eQTL results, gene function annotation and statistic analysis will not only more precisely identify candidate genes controlling complex traits and the expression of related genes, but also construct regulatory networks for these complex traits.

Abstract:In order to get insight into the biological function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD) , a yeast two-hybrid technology was carried out to screen the adult brain cDNA library using the full-length polyglutamine-expanded ataxin-3 as bait . Small ubiquitin-like modifier 1(SUMO-1) was identified as a novel ataxin-3-interacting protein. Subsequently, co-immunoprecipitation showed that both the wild-type ataxin-3 and the polyglutamine-expanded ataxin-3 were covalently modified by SUMO-1 in SH-SY5Y cell; immunofluorescence showed that the intranuclear aggregates formatted by the polyglutamine-expanded ataxin-3 co-localized with SUMO-1. Taken together, the data suggest that the biological function of ataxin-3 may be regulated by SUMO-1, and that SUMO-1 may participate in the pathogenesis of SCA3/MJD.

Abstract:The comparative studies of k-mer distribution in human and mouse TFBS sequences listed in TRANSFAC database are given. A non-alignment based approach for fast genome-wide discovery of transcription regulatory k-mer motifs (TRKMs) is proposed. The method is called distance-based conservative k-mer searching algorithm (DCKS) which is based on the conservation of k-mer pair distance. By use of DCKS the prediction accuracy of human transcription regulatory 7-mer motifs is: sensitivity 90%, specificity 78%, and correlation coefficient 0.65.

Abstract:The hippocampus plays a critical role during the consolidation of trace eyeblink conditioned responses (CRs). However, the role of its related structure such as dentate gyrus (DG) remains unclear. The present study was aimed at monitoring the activity of single granule cell in the DG during the consolidation of trace eyeblink CRs, and elucidating the possible role of DG during this hippocampus-dependent task. Guinea pigs (n=8) were trained on a trace eyeblink conditioning paradigm using a 200-ms tone conditioned stimulus (CS), a 200-ms corneal airpuff unconditioned stimulus (US) and a 600-ms trace interval. Controls consisted of pseudo- conditioned guinea pigs (n=8). Extracellular single unit recordings in vivo were performed in the DG of learner animals during the consolidation of trace eyeblink CRs. The results revealed that all the trace-conditioned animals acquired the trace eyeblink CRs over 14 training days, however, none of the pseudo-conditioned animals did. Furthermore, 23 of 40 single granule cells in the DG of learner animals exhibited heterogeneous activity patterns during the consolidation of trace eyeblink CRs such as increases in activities to the tone CS, trace interval or airpuff US. The results suggested that the DG might participate in the neural circuit important for the consolidation of trace eyeblink CRs, and that the granule cells might encode different information during the consolidation of trace eyeblink CRs.

Abstract:Several methods, including MTT assay, transmission electron-microscope (TEM), RT-PCR, flow cytometry, gene array and real time PCR, were used to investigate the reversal effect and potential mechanism of 4′-methylether-scutellarein from Verbena officinalis L.(VOL) on mutidrug resistance of JAR/VP16 cells. 4′-methylether-scutellarein produced a synergistic effect combining with the chemotherapeutics and reversed the resistant phenotype of JAR/VP16 cells. TEM analysis and flow cytometry confirmed apoptotic cell features, containing earlier apoptotic modifications, increased apoptotic cell percentage and typical apoptotic peak, when JAR/VP16 cells exposed to 4′-methylether-scutellarein. The gene array results showed that expression levels of some multidrug resistant genes, i.e. MDR1、MRP1、MRP2、MRP6、AHR、COMT and FGF2, were decreased. The expression levels of some apoptosis-promoting genes, i.e. Apaf-1、ASC、ATM、Bad、Bak、Bax and BimL, were increased, whereas those of some apoptosis-suppressing genes, i.e. Bcl-2、BFL1、NAIP and p63, were decreased upon 4′-methylether-scutellarein treatment. Sixteen genes were selected to be detected by real time PCR, and the results were identical with those of gene array. Taken together, 4′-methylether-scutellarein can efficiently reverse multidrug resistance in JAR/VP16 cells. The potential mechanism may be via inhibiting the multidrug resistant gene expressions and/or promoting cell apoptosis.

Abstract:Bone morphogenetic protein receptor-IB gene(BMPR-IB)which controls the fecundity of Booroola Merino ewes was studied as candidate genes on the high prolificacy in sheep. Seven sheep breeds, small tail han sheep and its crosses, Northeastern Half-fuzz sheep, Australian Merino sheep, German Mutton Merino sheep, Suffork sheep, Texel sheep and Charolais sheep, were used. Single nucleotide polymorphism of BMPR-IB gene was detected in all sheep breeds by PCR-RFLP. The results showed that there was a same A746G mutation in BMPR-IB gene as that of Booroola Merino ewe, the frequency of B allele in the small tail han sheep and its crosses was significantly higher than that of the other two breeds, and the same mutation did not exist in other four breeds. The sheep carrying the B allele were able to ovulate more ova than non-gene carriers, and ovulating at a smaller diameter. The pregnant rates with respect to frozen embryo were 38.78%, 45.71% and 66.67% for the ++, B+ and BB animals, respectively. However, the results of present study showed that the A/G mutation in BMPR-IB gene appeared to positively affect both ova number and pregnancy process, which suggests that BMPR-IB gene play an important role in the regulation of sheep prolificacy. So ideal genotype sheep by PCR-RFLP as recipient is selected. It provides a new approach to improve pregnancy rate of embryo transfer.

Abstract:Using Western blotting technique, the effects of auditory deprivation and tone exposure on the relative expression level (REL) of NMDA receptor subunit NR2B protein of rat auditory cortex during early postnatal days were studied. The results showed that the NR2B protein expression of PND14 and PND28 decreased significantly (P < 0.05, P < 0.01) when auditory deprivation for 7 days. Tone exposure after auditory deprivation for 7 days made NR2B protein level increased significantly (P < 0.05). It showed bidirectional regulation of NMDA receptor subunit NB2B protein. No significant effects of auditory deprivation and tone exposure on the relative expression level of NR2B protein of PND42 (P > 0.05) were found. The results indicated that, auditory deprivation and experience can modify the protein expression level of NMDA receptor subunit NR2B during the critical period of rat postnatal development. The findings provided important data to the study of the mechanisms for the developmental plasticity of sensory functions.

Abstract:GIP, the acronym from gastric inhibitory peptide or glucose-dependent insulinotropic polypeptide, which is a kind of gastrointestinal regulatory peptide composed of 42 amino acids, plays an important role in stimulating insulin releasing in the pancreas,inhibiting gastric acid secretion in the stomach, as well as inducing progenitor cell proliferation in the brain. The application of GIP is promising in clinic. It is difficult and costly to get GIP with chemical extraction method, and it can't be produced cosmically, so it is valuable and significant to produce recombinant GIP by genetics and test its bioactivity. The cDNA sequence coding for human GIP mature peptide and composing of preferred codons of E. coli. was synthesized, and was expressed in prokaryotic expression pET32a(+) system. The recombinant E. coli was fermented in a small scale and the expression condition was optimized. The expressed recombinant human GIP (rhGIP) was purified by affinity method. The bioactivity of the purified rhGIP was tested through its effect on inhibiting gastric acid secretion and decreasing blood glucose concentration in SD rats. The effect on PC12 cell producing NO free radical was assessed through morphology observation and the NO concentration test. A PC12 cell injury model was constructed by adding Aβ_25-35 into the culture medium, different dose of rhGIP was added into this model, and the activity of PC12 was tested by MTT (2-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide) staining. The results suggest that hGIP gene is cloned successfully, the recombinant protein constitutes 35% of the total protein of the cells, some of which was soluble, while the others exist as inclusion body. The relative molecular mass of the recombinant protein is 26 ku as expected. The purified rhGIP showed immunoreactivity. The optimal condition for inducing to expression is with the cell concentration of 0.5 in A₆₀₀ value, the IPTG concentration of 0.5 mmol/L, under 37℃ and induced for 4 h. The cell lysis supernatant was purified with immobilized metal affinity chromatograph. The production of soluble rhGIP is about 1.2 mg/L culture medium, and the purity is 85%. Purified rhGIP significantly inhibited the secretion of gastric acid and raised the pH value comparing with NC group in SD rats (P < 0.05). There was no significant difference between the rhGIP and standard GIP group (P 0.05).After injection of rhGIP for 15 min, the concentration of blood glucose in plasma decreased remarkably comparing with control group in a high blood glucose environment (P < 0.05). In the later 30 min, there was no remarkable difference (P > 0.05). Also, there was no difference between rhGIP and standard GIP group (P > 0.05).In the research to test the effect of rhGIP on cell nutrition and PC12 cell to avoid neural injury and anoxic injury, it was found that the level of NO was significantly decreased (P < 0.01), the survival rate of the PC12 cells was increased, and neurite extending was better. The activity of the high and medium rhGIP concentration group is higher than that of neural injury group and anoxic injury group (P < 0.05), and the activity is increased as dose-dependence. The differences among rhGIP, GIP and NC group is not remarkable (P > 0.05). The results showed that the rhGIP has been successfully expressed and purified and showed obvious immunoactivity. The recombinant product was shown to have a wide spectrum of biological activity, such as inhibiting gastric acid secretion in the rats stomach, decreasing the concentration of blood sugar in plasma, as well as nutrition and protection to neural cells.

Abstract:Interferons (IFNs) play important physiological roles in early embryo development, but its mechanism is still unclear. The full length cDNA of interferon responsive gene (IFRG) was cloned in rabbit ovary (Accession No. AJ584672). The expression profile of IFRG in oocytes and preimplantation embryos was detected using RT-PCR. IFRG was expressed in oocytes and preimplantation embryos in rabbit, which provided some clues for better understanding the role of IFNs during embryo development. In situ hybridization showed that IFRG was highly expressed in granulosa, thecal and cumulus cells, which were consanguineous interrelation to follicles development. So it was proved that IFRG expression was involved in the follicles development, maturation and ovulation in rabbit.

Abstract:In order to construct an efficient and safe engineering vaccine against E. tenella, the relative gene of Rhomboid protein family of E. tenella F2 hybrid strain was amplified by RT-PCR technology and subcloned into the downstream of complex promoter of fowlpox virus vector pUTA2, recombinant fowlpox virus transfer vector was constructed successfully. Plasmid of fowlpox recombinant virus and fowlpox virus were co-transfected into chicken embryo fibroblast (CEF) and identified by BrdU drug, RT-PCR and Western blotting respectively. The result showed that the E. tenella fowlpox recombinant virus (rFPV-Rhomboid) was obtained. Furthermore, rFPV-Rhomboid was amplified in CEF, then chicken was immuned with rFPV-Rhomboid and the indication of immunity was detected. The result indicated that the contents of CD4⁺ and CD8⁺ in peripheral blood were significantly higher than that in control group (P<0.05). The weight gained of chicken vaccinated rFPV-Rhomboid was different from the chickens of control group significantly (P < 0.05). The recombinant protein had immunogenicity and could induced strong immune response and immune protection ability.

Abstract:A proteomic approach was used to analyze differential expression of proteins during soybean (Glycine max) N2899 seed germination at specific stages of 0 h, 8 h, 36 h and 60 h after imbibition. The results show that on the 2-DE gels stained by coomassie brilliant blue, PDQuest image software detected about 350 protein spots, of which 24 spots show more than 2.5-fold changes in abundance, and most of soybean seed storage proteins weren't mobilized during seed germination. At the first stage of germination, 10 proteins show changes in abundance. At the second stage, the quantities and categories of differentially expressed proteins increased and the abundance of 15 proteins dynamically changed. During radicle protrusion seed coat, the abundance of 14 proteins reached a peak. These suggested that the internal metabolisms of imbibition seeds became more active. These 24 proteins treated by tryptic in-gel digestion were characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprintings of all were obtained. Soybean UniGene database was used to identify proteins, of which 6 proteins were identified. They were nucleotide diphosphate kinase, proglycinin A_1aB_1b subunit, thioredoxin fold, 35 ku seed maturation protein, heat shock protein and seed maturation protein PM36. The potential functions of these proteins during seed germination were discussed.

Abstract:Glechoma hederacea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.

Abstract:Miniaturization of analytical instrument is one way of addressing the issues of sensitivity, measurement speed, throughput and cost of analysis in biology analysis. Microfluidic devices can be used to manipulate the microscale sample expediently, and have many advantages including minimum sample consumption and minimum cross contaminant, which are typical problems in other conventional standard fluidic devices. The highly integrated microfluidic devices were suitable for high-density, parallel sample processing, and high-throughput analyses with extremely high duty cycles. Besides the optical spectroscopic measurement and electrochemical detection, mass spectrometry has been coupled to microfluidic devices as detector, resulting in rapid analysis of complex biological samples with high throughput and confidence. Microfluidic devices utilizing chromatographic or capillary electrophoresis separation techniques are under fast development, showing a predominant trend in modern analytical science. After giving a brief introduction to background of the microfluidic devices and fabrication techniques, documenting the technologies and applications of microfluidic mass spectrometry for the analysis of biological samples and emphasizing on the emergence of interfaces coupling microfluidic devices to various mass spectrometers for applications in proteomics, metabolomics and other biotechnology areas are reviewed.