Abstract:For a long time, proteins have been considered as the expression forms of DNA or RNA and proteins alone cannot store and transmit biological information. But storing and transmitting biological information are two essential characteristics of genetic materials. With the appearance and development of prion biology, people have known that proteins alone have the ability of storing and transmitting biological information. In some sense, proteins are also genetic materials. It is very necessary to rediscover and reinterpret prion biology in light of this new concept. Common rules of protein-based storage and transmission of biological information and the diversity of their manifestation can be seen by reviewing the history of mammalian prion biology and fungal prion biology and by introducing the latest progress in this field.

Abstract:MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18~25 nt in eukaryotic organisms, which can regulate the complementary mRNAs at the post-transcriptional level through cleavage or translational repression of the mRNA targets. Recent years, several hundred miRNAs from animals and plants have been identified. These small modulatory RNAs are cleavaged from a precursor of 60~200 nt RNA hairpin. In animals, the primary transcripts of miRNA genes (pri-miRNAs) are recognized and cleaved into precursor miRNAs (pre-miRNAs) soon by an RNase Ⅲ family nuclease, Drosha; then, pre-miRNAs are transported from the nucleus to the cytoplasm. Once in the cytoplasm, pre-miRNAs are recognized and processed into their mature form by another RNase Ⅲ , Dicer. The procedure is briefly summarized, and the biogenesis of plant miRNAs is also discussed. Further research on the pathway of miRNA maturation can help us to know the mechanism of these small RNAs acted as important regulators, and can investigate their critical roles during development and disease.

Abstract:Mediator, originally discovered in yeast and composed of multi-subunits with a high molecular mass , is an essential component of the RNA polymerase Ⅱ general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. Regulatory information could be conveyed through changes in Mediator conformation that would influence the transcription initiation process. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.

Abstract:The role of the leucine zipper domain in actin binding of p57 was investigated by the methods of the F-actin co-sedimentation studies in vitro, the immunofluorescence co-localization analyses in cells, the Western blot and density scan assays. The results showed that the leucine zipper-containing domain alone has no actin-binding activity, while the C-terminal deletion mutation removing this domain or the point mutation destroying leucine zipper domain leads to great reduction of actin-binding activity of p57. Furthermore, the degrees of degradation in actin-binding activity caused by the two mutations were assessed in quasi-quantitative analysis in vitro and in cells and both mutations exhibited the extremely similar depressing effects. These data strongly suggest that the leucine zipper domain is important for actin binding of p57.

Abstract:To investigate the protection effect of DNA vaccine in mammalian and avian systems, the DNA vaccine was inoculated in both BALB/c mice and SPF chickens immunized with DNA vaccines encoding hemagglutinin (HA) from A/Goose/GuangDong/1/96 (H5N1) virus. The mice and chickens were immunized twice, 3 weeks apart, by electroporation into muscles or intramuscular injection. Two weeks after the second immunization, the mice and chickens were challenged with a lethal dose of homologous virus. The mice and chickens immunized by electroporation obtained completely protection against the virus, and could effectively inhibited viruses to replicating in mouse lung and chicken cloaca. At the same time, these protections were companied by high levels specific antibody to H5N1 AIV, while the blank plasmid controls experience 100 percent mortality following challenge. Furthermore, in the experiment of mice by eletroporation, stronger obviously CTL activity were observed after challenge. Thus, the cellular immune responses of the mice immunized by electroporation were exhibited. These results strongly demonstrate that HA DNA vaccines provide effective protection against influenza virus infection in mammalian and avian, and suggest that electroporation is one of the efficient gene delivery systems for the transfer of influenza DNA vaccine in both humoral immunity and cellular immunity.

Abstract:Phosphoinositide3-kinase (PI3K) is involved in regulation of many kinds of physiological processes in cells, such as vesicle transportation, cytoskeleton reorganization, cell survival, phagocytosis, apoptosis, and so on. It is an important regulator of vesicle edocytosis. According to preview reports, PI3K seems to be one of regulators of cell secretion. To inspect this, the effect of PI3K on PC12 cell secretion was checked by using its specific inhibitor wortmannin. Wortmannin inhibited PI3K activity indicated by loss of EGFP-2xFYVE fusion protein binding to the PtdIns-3-P which is localized in early endosome, and did not change PC12 cell response to flash including its kinetics and calcium dependence. The result demonstrated that PI3K has no effect on PC12 cell secretion itself, but does not exclude the possibility that it enhances cell to response to repetitive strong stimuli by its accelerating effect on vesicle endocytosis which speeds up the refilling of releasable vesicle.

Abstract:Fhx/P25 in silkworm, Bombyx mori, one of the main components of silk fibroin, is presumed in previous reports to be expressed exclusively in the posterior silk gland (PSG) of the animal with strict territorial and developmental specificities. On the basis of a large-scale analysis of the silkworm EST data, it was found that Fhx/P25 gene is transcribed not only in the posterior silk gland, but in the ovary and in other tissues of the larvae at day 3 of the fifth-instar as well and that this gene has distinct transcription start sites (TSSs) in the posterior silk gland and the ovary. The TSS in the ovary is located about 115 bp upstream sequence of that in the posterior silk gland. Subsequent RT-PCR, FQ-PCR and sequencing have verified the validity of this presumption. In addition, alternative splicing is predicted in pre-mRNA of Fhx/P25 gene and confirmed by RT-PCR. In conclusion, Fhx/P25 gene is not a gene with strictly tissue-specific transcription. Complicated regulation mechanisms may exist for its transcription and expression and it may have other functions to perform.

Abstract:Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays. The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amount for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.

Abstract:Endolysins or lysins are cell-wall-hydrolysing enzymes synthesized during late gene expression in the lytic cycle of bacteriophage mutiplication and enable the release of progeny virions from infected cells through degradation of the bacterial peptidoglycan. The gene10 was amplified from purified DNA of Mycobacteriophage D29 by using PCR and was expressed with 6×His tag in E.coli. The soluble portion of recombinant protein was purified by Ni-NTA column and the activity was detected. The results showed that gp10 is a bi-functional enzyme, has chitinase and lysozyme activities. The cell wall degradation was observed by SEM.

Abstract:To show the flexible adapter design of restriction display (RD) technique in constructing peptide library, two series of adapters were designed according to vector pET22b in E.coli and vector pNMT-TOPO in S.pombe. Each series of adapter has three in turn one-base-increment adapters, which allowed the inserted DNA fragment probably expressed in the correct reading frame. HIV-1 subtype B whole gene was used as an example, and fragments expression libraries with two different adapters were constructed by RD technique, randomly 12 clones from each library were sequenced for translation analysis. As a result, a clone from the prokaryotes library was obtained, which could encode HIV Pol peptide. Then the positive plasmid was induced to express protein in E.coli BL21(DE3). SDS-PAGE and Western blot showed positive result, which is consistent with expected. It can be concluded that RD technique is a new approach for constructing genome random peptide library, and its flexible adapters design can meet with kinds of expression vectors.

Abstract:Combined DNA vaccine encoding Ag85B, MPT64 and MPT83 of Mycobacterium tuberculosis were formulated into DDA and MPL to immunize mice and then the immunogenicity and protective efficacy of each group were evaluated. The DDA and MPL groups induced a much more enhanced Th1-type cellular response indicated by the higher levels of IFN-γ compared with that without any adjuvant. In DDA group, antigens specific IFN-γ for Ag85B, MPT64, MPT83 are (265.37±79.2) U/ml ， (185.31±58.3) U/ml ， (108.13±54.4) U/ml respectively which are 16 U/ml ， 45 U/ml ， 2 U/ml higher than that of the non-adjuvant group. The bacterial CFU in lungs and spleens of the DDA group was reduced 1/5 and 1/4 respectively relative to the same combined vaccine with MPL and without adjubvants. The pathological lungs slices of adjuvant groups gave consistent result that showed less damage than non-adjuvant group due to influx of epithelioid macrophages and less neutrophils. In conclusion, DDA is more efficacious than MPL as adjuvants to enhance immune efficacy of combined DNA vaccine in mice.

Abstract:P15RS was identified as a novel gene cloned from the human melanoma cell model MLIK6 overexpressing p15INK4b. P15RS has been reported as a negative regulator of cell proliferation in G1 phase. To investigate the localization of P15RS, EGFP-P15RS, a fusion protein with EGFP reporter was constructed and overexpressed in BGC-823 cells. The results showed that EGFP-P15RS protein was distributed in nuclear in interphase (G1, S, G2 phase) of BGC-823 cells. The EGFP-P15RS protein was observed not to localize in chromosome in M phase, suggesting that P15RS might be located in karyoplasm.

Abstract:NOR1 is one of the candidate tumor suppressor genes associated with nasopharyngeal carcinoma (NPC). The mammal expression vector of NOR1, pcDNA3.1(+)/NOR1 was constructed and was introduced into HNE1 cell to explore the effect of NOR1 gene on HNE1. The integration of the exogenous vector DNA and the reexpression of NOR1 were detected by RT-PCR and Northern blot respectively. Finally, the cytobiological characterization of positive clone was analyzed by cell growth curves analysis, soft agar assay, cytometry. The growth of HNE1 cells transfected with NOR1 gene was dramatically inhibited compared with the parent HNE1 cells. Flow cytometric data showed that more NOR1 transfected cells went into G0/G1 phase than controls, and it also presented decreased clonogenicity in soft agar. The research indicated that the NOR1 could play a critical role in the progression of NPC due to its results providing a basis study of the function of it.

Abstract:Sam68, a nuclear RNA binding protein, is the Src mitotic target and specifically tyrosine phosphorylated during mitosis. It has also been demonstrated to associate with various signal transduction molecules, thereby raising the possibility of its role in cell cycle control as a modulator of the signal transduction and activation of RNA metabolism. To elucidate the physiological function, a Sam68-deficient cell line was isolated from the chicken DT40 cell line by gene disruption. The Sam68 deficient cells exhibited markedly decreased growth, and forced expression of chicken Sam68 cDNA in the mutant cells restored the cell growth. Cell cycle analysis revealed that the growth retardation was due to elongation of the G2-M phase, however, the kinase activity associated with Cdc2 remained unaltered. The results indicate that Sam68 may play a critical role in G2-M progression in a manner independent of the control of cyclin/Cdc2 kinase activity.

Abstract:Choosing 8~10 days cultured hippocampal neurons of SD rat, using Calcium Orange AM and DAF-FM diacetate as the fluorescent indicators of intracellular Ca2+ and nitric oxide(NO), simultaneous detection of intracellular Ca2+ and NO was proposed by double-label method on laser scanning confocal microscope (LSCM). The dyeing process includes two steps and “Two Track” mode of LSCM is applied to realize simultaneous detection of intracellular Ca2+ and NO through quickly switching excitation wavelengths. The experiment results show that there is no cross talk between two dyes and the double-label method can reveal the changes of intracellular Ca2+ and NO concentrations under the stimulation of N-methyl-D-aspartate (NMDA), quite consistent with the results of respective single-label experiments. The analysis of slicing image sequences of double-labeled neurons shows that both Ca2+ and NO are mainly located in the center area of cell bodies, while their distribution details are different. The results suggest that the double-label method can simultaneously detect the intracellular Ca2+ and NO in cultured hippocampal neurons and thus provide an approach to investigate the roles of Ca2+ and NO in neurons as well as the interaction between them.

Abstract:Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein. Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological function of this plant PHGPx.