Abstract:The mechanisms of regulating stem cells self-renewal is one of the most important issues in stem cell biology. Haematopoietic stem cells (HSCs) have the ability to renew themselves and to differentiate into all lineages of the blood but, the mechanism that control HSCs self-renewal remain unclear. Most researches showed it was controled by the signal and growth factors from the microenvironment and the cell-autonomous components. The wnt signaling pathway plays an important role in this process. The HSCs self-renewal, the mechanism, the role of wnt signaling pathway and the perspectives of application and research in this field are discussed.

Abstract:Synaptic plasticity is a physiologically important mechanism underlying neuronal information processing. In terms of expression site, synaptic plasticity can be divided into presynaptic and postsynaptic. Presynaptic plasticity is implicated in the modulation of the neurotransmitter release machinery and consequently in synaptic strength. From a physiological perspective, this type of plasticity could be derived from a change in quantal size, active zone structure, probability of transmitter release, especially, synaptic vesicle dynamics: from synaptic vesicle trafficking to the nerve terminal, docking at or fusion with the presynaptic plasma membrane, and finally, reconstitution following endocytosis. Each of these steps is mediated by the concerted activities of multiple proteins and protein complexes, thus presenting numerous points at which the cascades leading to effective neurotransmitter release could be modulated. Potential mechanisms by which the synaptic vesicle release could be modulated and synaptic activity could be silenced or enhanced at the presynaptic terminal are reviewed.

Abstract:RNAi (RNA Interference) has been recently found as an innate cellular process activated when a double-strand RNA (dsRNA) molecule enters the cell causing the degradation of RNAs of identical or closely similar sequences, including mRNAs. It would suppress the expression of specific genes. It proves to be a more efficient tool than the antisence method with more specificity and now it is the focus of researchers allover the world. People are trying to find ways to make RNAi of an efficient clinical method but they also found that it is a long way to go before it becomes successful at last. To make an analysis of the problems and solutions during the process based on the fundamental knowledge that were already known are reviewed.

Abstract:Human neuronal tau aggregates by incubation at 37℃ (pH 7.2) and the aggregated tau is inactivated in promoting tubulin assembly. Furthermore, aggregated tau induces inactivation and conformational changes of rabbit muscle lactate dehydrogenase, although native tau can promote LDH activity. It suggests that tau aggregation may affect the energy metabolism in which LDH is involved.

Abstract:Some of proteins associated with differentiation and development of cells and tissues contain zinc finger domain. To clone and characterize proteins that are related to the hemopoietic cell differentiation and development. C2H2 zinc finger domains were amplified by RT-PCR with degenerated primers designed according to the conserved amino acids. Total RNA was extracted from human bone marrow. Some expressed sequence tags (ESTs) containing the zinc finger motif were acquired and one of them was used as probe to screen human bone marrow cDNA library. As a result, a new gene (GenBank accession number: AF246126) was cloned and designated HZF2. The full length of the cDNA is 3 888 bp and the open reading frame encodes a protein with 686 amino acid residues containing 17 typical and 2 atypical C2H2 zinc finger motifs. Northern blot and Human RNA Master Blot analysis suggested that HZF2 might play an important role in T cell development and amplification. The fragment encoding the complete HZF2 peptide was inserted into the eukaryotic expression vector pEGFP-N1 and introduced into 3T3 cells. The fusion protein was located in cell nuclei, which was a consistent with the conjecture that HZF2 may function as a DNA-binding protein to regulate gene transcription.

Abstract:A gene trap construct was used to transfect mouse ES cells. 36 ES colonies trapped by one copy of neo gene were obtained. The β-galactosidase activity was detectable in 14 ES colonies. ES cells from 3 trapped lines were introduced into blastocysts by microinjection. Two chimeric mice lines were generated. One trapped mutation went through the germline. Genomic sequences adjacent to integration sites of the constructs were isolated by plasmid rescue. The results of sequencing suggested that the trapped gene is possibly a novel gene. The expression pattern of this gene shown by β-galactosidase expression was restricted in abdomen and lib bud of E10.5 mouse embryo.

Abstract:The UV absorbance ratio(A₃₉₂/A₂₈₀) was developed to survey the purity of the P450cam mutant protein during the purification of the mutants protein. Thus the course of the purification became compact and the efficiency of the purification had been improved. β-Mercaptoethenol had been developed to maintain the deoxidization of the protein during the purification. Crystals of the F87L/Y96F/L244A/V247A mutant were grown by vapor diffusion method. X-ray diffraction data were collected to 0.22 nm resolution on a Mar Research area detector in house. The structure was determined by Difference Fourier Method. The final crystallographic R factor and R_free factor are 0.197 and 0.247 respectively. The RMS deviations of bond length and angle of the mutants are 0.001 77 nm and 1.96°respectively. Structure comparison indicates that there is no obvious conformaional change between P450cam and the F87L/Y96F/L244A/V247A mutant. After mutation, the structure of the active pocket became larger but its hydrophobility increased, which are consistent with the aim of mutantion

Abstract:BPOZ is one protein containing ankyrin repeat and BTB (POZ) domain which gene is located at 3q21.3 in human genome. It was found that the expression of BPOZ gene is down-regulated in human ovary tumors, suggesting it is potentially a cell growth or tumor suppressor gene. BPOZ gene knockout mouse model was established for further in vivo study of its normal function and the role of BPOZ in tumorigenesis. The mouse genomic DNA sequence of BPOZ gene was verified through bioinformatic analysis. According to the BPOZ genomic DNA sequence, the strategy of gene targeting and constructing of knockout vector (XpPNT-BPOZ) were established to delete the region of mouse genome, which spans from exon 2 to 12 of BPOZ gene. The gene knockout vector XpPNT-BPOZ was constructed and confirmed by restriction enzyme digestion and sequencing. Electroporation of ES cells with XpPNT-BPOZ and screening of both G418 and Ganciclovoir resistant clones were performed according to common protocol. The homologous recombined ES cell clones were identified by PCR and confirmed by Southern blot analysis. After transplantation of homologous recombined ES cells into blastocysts through microinjection and mating of chimeras with C57BL/6J mice, 30 offspring with Aguoti fur in color were acquired. 15 of them (50%) show genotype heterozygous for BPOZ. The BPOZ heterozygotes were intercrossed to generate mutants homozygous. Finally, the cohort of mutants homozygous for BPOZ was established. The mutants are fertile, and further phenotype analysis is under way.

Abstract:The colleterial gland in the silkworm (Bombyx mori) grew gradually until 2 days before emergence, and markedly enlarged due to the accumulation of glue like substances (mainly including 85% water and 11% proteins). However, the Ng mutant female moth only secreted a small amount of glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis was used to screen the proteins pattern between normal and Ng mutant colleterial gland to find quantitative and qualitative difference in proteins expression. Protein spots were resolved in the secretory region of colleterial gland of silkworm and more than 700 protein spots were resolved and most of the proteins were distributed in the area from 30 ku to 70 ku and pH 4～8. Through the comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage and moth stage. However, these proeins have no expression in the Ng mutant especially spot No.2 and 3. These differentially expressed proteins were actin identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results indicated that the actins participate or regulate the exocytosis of colleterial gland and other differential expressed proteins might be having some relations with the glue-like proteins secretion.

Abstract:Although p53 gene mutation is a rare event in human nasopharyngeal carcinoma (NPC), over-expressed/accumulated p53 protein is dysfunction in most of NPC. However, till now the mechanism of p53 protein inactivation remains unclear. In order to elucidate the mechanisms of p53 inactivation, p53 interacting proteins in the human NPC HNE1 and HNE2 cells were separeted and identified. p53 binding proteins were recovered by anti-p53 antibody immunoprecipitation with the total proteins from NPC HNE1 and HNE2 cells respectively. The recovered protein complexes were subjected to SDS-PAGE. The 5 separated protein bands were cut from the gel, in-gel digested and analyzed by LC-ESI-MS/MS. Then proteins were identified by peptide sequence tags(PST) and database searching, and were confirmed by immunoprecipitation and Western blot analysis. The results show that nine p53 binding proteins were identified, which were GRP-78 and GRP-75 of HSP 70 family members, GRP-94 of HSP 90 family members, laminA/C, Alpha-actinin 4, Ezrin/Cytovillin, DNA replication licensing factor/MCM3 protein, CD98/4F2 heavy chain and protein kinase C. It can be concluded that this study first time identified nine p53 binding proteins in NPC, which provide important clues to elucidate the mechanism of p53 over-expression and inactivation in NPC.

Abstract:Ordered differential display (ODD) was used to clone differentially expressed genes in rice induced by Magnaporthe grisea. Of 37 cDNA clones initially identified by reverse Northern blot, 5 clones were chosen as probes for further Northern test. The result showed that all of these 5 clones were expressed both in highly, moderately resistant and susceptible rice lines in a pathogen inducible manner. According to the homology analysis, these clones were suggested to be involved in plant defense response by: (1) inhibiting growth of fungus and even causing its death; (2) excluding toxic compounds delivered by fungus; (3) priming signal transduction; (4) and/or regulating metabolic activities in agreement with plant defense.

Abstract:In order to study the interactions between plant virus movement protein and pectin methylesterase (PME), PME gene from tobacco genome was cloned using RT-PCR method and the sequence was determined (GenBank accession No.AY238968). Sequence analysis showed that there were two conserved domains in PME protein: PMEI and pectinesterase. Multiple copies of PME gene were detected in tobacco genome through Southern blot analysis. Western blot result indicated that two types of PME protein existed in tobacco. But Northern hybridization detected only a full length transcript of PME, which further identified that there was post-translational process. Yeast two-hybrid result demonstrated that PME could not interact with the identified movement protein of rice dwarf virus (RDV), Pns6, but does interact with Pns11, a nucleic acid binding protein of RDV. It implies Pns11 may participate in the movement of RDV.

Abstract:Two new type Ⅱ ribosome-inactivating proteins (RIPs) were isolated from the mature seed of camphor tree (Cinnamomum camphora). They are named cinnamomin and cinphorin. The molecular mass of cinphorin A-chain is only half of cinnamomin A-chain, while their B-chains are the same. Their A-chains are RNA N-glycosidases, and the B-chains are lectins. The intrinsic cytotoxicity of type Ⅱ RIP is greatly dependent on the carbohydrate-binding activity and the specificity of its B-chain. The lectin activity of cinnamomin and cinphorin are investigated and compared with each other. They showed similar hemagglutination activity. Their saccharide binding specificities were studied by hapten inhibition, indicating that they were both galactose-specific. However, N-acetylgalactosamine failed to bind to the two RIPs as ricin/abrin did. The interactions of the two RIPs with specific saccharides were also investigated by fluorescence spectroscopy through which association constants were obtained. Their association constants of galactose or lactose were found to be identical.

Abstract:His-tagged Chinese hamster dihydrofolate reductase (DHFR) expression in pET vector system has been reported very low. A newly modified pET protein expression vector, pET-DB, was utilized to overexpress of it in soluble form. The amount of DHFR reaches to 46% of the total protein in E.coli cells. This His-tagged DHFR could be purified routinely by Ni-NTA agarose resin and the His-tag could be removed by thrombin easily. This engineered DHFR has the same enzyme activity as the enzyme without His-tag obtained by iso-electrophoresis.

Abstract:Nuclear transfer has been used to clone several mammalian species，including sheep, goats, cattle, pigs, mice, rabbits, houses, and mules. However, still there are many problems such as low efficiency, high abortion, malformation, huge fetus, high neonatal death need to settle. So it was important to analyze a wide variety of genes in individual cloned embryos. But using conventional RT-PCR strategies permitted analysis of only a few genes in one embryo. A global RT-PCR strategy has been used, allowing the analysis of almost hundreds of genes from a single embryo. This method was not seen used to analyze the genes in cloned embryos. Bovine oocytes have been used to investigate the expression of genes related to development such as IL6, IFτ, CX43, PSMC3, oct4, DNMT1. The results were same as those obtained by using conventional RT-PCR strategies. Further it forth the first time reported the expression of DNMT1 and PSMC3 gene in bovine oocytes.

Abstract:The microrheological characteristics of reticulocytes in vivo was studied. Method of anemia in rabbits induced by ⁶⁰Co radiation was used. The measurements of percent of reticulocytes, deformation index, orientation index, etc in the processes of reticulocytes changing into erythrocytes in vivo for 72 h were performed. It was shown that there were obvious changes in the reticulocytes rheological characteristics in the courses of this changing. Therefore, the found had some basic theoretical and clinical significance.