Abstract:Aminoacyl-tRNA synthetase is a class of ancient proteins, catalyzing the first reaction of protein biosynthesis. It has been found that they also participate in a lot of other cellular processes such as editing, tRNA maturation and transfer, RNA cleavage and function as cellular factors. Recent studies showed that some mitochondrial aminoacyl-tRNA synthetases are closely related with human diseases. A single point mutation in intervening sequence 2 (IVS2) of human mitochondrial arginyl-tRNA synthetase gene causes abnormal cleavage of its transcript, resulting in pontocerebellar hypoplasia. A series of mutations in human mitochondrial aspartyl-tRNA synthetase gene cause rapid decay of its mRNA or alteration in protein primary sequence, leading to leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. A single nucleotide polymorphism in human mitochondrial leucyl-tRNA synthetase is significantly associated with type 2 diabetes. These results further enhance our understanding about the cellular function of aminoacyl-tRNA synthetase and promote studies toward the mechanism and therapy of aminoacyl-tRNA synthetase-causing mitochondrial diseases.

Abstract:TMPRSS3 (transmembrane protease, serine 3) is a member of Ⅱ transmembrane serine proteases (TTSPs), and like the other members of this family, it contains typical domains including a serine protease domain, a transmembrane domain, a LDL receptor-like domain (LDLRA), and a scavenger receptor cysteine-rich domain (SRCR). Four alternative protein isoforms have been described, and isoform A is thought to be primary isoform which is expressed in many tissues, especially in the cochlea. TMPRSS3 protein is primarily localized in the endoplasmic reticulum membranes where it may be anchored by its transmembrane domain. TMPRSS3 is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). Therefore TMPRSS3 is thought to be involved in the development and maintenance of the inner ear, and isoform D may be proposed as a novel diagnostic marker in ovarian carcinoma. TMPRSS3 protein is the first protease which mutation could lead to deafness. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage. However, it is not clear about TMPRSS3 substrates and its function. The epithelial amiloride-sensitive sodium channel (ENaC) which is regulated by membrane-bound channel activating serine proteases (CAPs), a member of TTSPs, may be a potential substrate of TMPRSS3, but this hypothesis is still to be verified in vivo. With the development of protease research and the application of protease proteomics, substrate degradomes of a protease may therefore represent an important tool for the research of TMPRSS3 function and its molecular mechanism.

Abstract:For the characteristics of numerous subtypes and frequent variations of influenza virus, it brings great difficulty to influenza in the prevention and control. Thus, the most pressing matter of the moment is to reinforce the research in pathogenesis and transmission mechanism, as well as to develop new type vaccines. Recently, the development of reverse genetics technique breaks a new path for influenza virus in research of function analysis and preparation of vaccines.

Abstract:Numerous inherited diseases are found to result from gene mutations that lead to mutant proteins, which can not fold correctly, fail to undergo trafficking, and are unable to reach their sites of action. Recently, pharmacological chaperones are developed as new therapeutics to rescue proteins defective in folding and trafficking. They are small cell-permeable chemicals, such as substrates, receptor ligands and enzyme inhibitors, which selectively recognize mutant proteins in the endoplasmic reticulum, correct their folding, stabilize the conformation, facilitate their trafficking to destination and yield functional proteins directly. Pharmacological chaperones represent promising avenues for the treatment of endocrine and metabolic diseases. They rescue a number of mutant proteins destined for the plasma membrane or organelles, such as ABC transporters, G-protein-coupled receptors, lysosomal enzymes and so on. Currently, one pharmacological chaperone has been successfully tested in clinical studies, and a lot of pharmacological chaperones have been tested and displayed positive results in cellular system and animal. This indicates that pharmacological chaperones will have great potential and broad prospects for clinical application in the future.

Abstract:The differentiation of neural stem cells/neural precursors (NSCs/NPs) is a hot spot in neurobiological research. It used to identify their differentiation degree only by morphologic appearances. The functional characteristics, such as electrical properties of cellular membrane and ion channel activities, are drawing more and more attention with the development of patch clamp technique. It was summarized the recent progress in the study of NSCs/NPs' functional differentiation using patch clamp, some existing problems and research perspectives were suggested.

Abstract:The Ca²⁺ wave is a chain reaction of intracellular Ca²⁺ release channels through a Ca²⁺-induced Ca²⁺ release mechanism. In cardiac myocytes, Ca²⁺ wave has drawn much attention because it is found to induce arrhythmia genesis. To investigate the microscopic process of wave propagation, Ca²⁺ imaging was performed with high spatial and temporal resolution via a laser-scanning confocal microscope combined with loose-seal patch clamp. These observation and analysis revealed that Ca²⁺ waves originated from a stochastic recruiting of Ca²⁺ release units (CRUs) by a pioneer Ca²⁺ spark, which had a low possibility in normal cells. During wave propagation, the ‘waiting’ time that the wave propagate between two neighboring CRUs along propagation direction distributed normally, and cells with a lower speed had a more dispersive distribution of ‘waiting’ time. To study the cause of the randomicity, the wave propagation was simulated with a numerical model. The simulation showed that the intrinsic stochastic open process of CRUs can fully explain the above phenomenon. Increasing the maximal open probability of CRUs reduced the randomness of wavefront propagation and enhanced the average velocity of wave meantime. These experimental and numerical results provided an unequivocal quantification for the stochastic behavior of wave initiation and propagation.

Abstract:Gene targeting in livestock fibroblasts has proven extremely difficult to achieve, particularly on silent gene locus. To obtain IgH functional disruption goats used for humanized antibody research, the goat IgH gene, which is transcriptionally silent in fibroblast, was knocked out and the targeted fibroblasts can be used to produce goats with IgH disruption through somatic cell clone. The goat immunoglobulin heavy chain J-Cμ fragment was amplified from the genomic DNA of goat fetal fibroblasts GEF88 through PCR and used as homologous arm to construct an isogenic Positive-Negative Selective targeting vector GTIgH. Then the GEF88 were transfected with the linearized targeting vector GTIgH through electroporation and selected in cell culture medium with 0.8 mg/L puromycin. 1 of the 362 drug-resistant clones was positive for targeting events through PCR screen, and the targeted clone was further confirmed by sequencing and Southern blotting. This suggests that one allele of IgH gene has been successfully knocked out in goat fetal fibroblasts.

Abstract:Mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) can not only support hematopoietic stem cells (HSCs) expansion in vitro as stromal cells, but also alleviate complications and accelerate the recovery of hematopoiesis during hematopoietic stem cell transplantation. The ratio of successful isolation and culture of UCB-MSCs, however is only about 20%～30% to date. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In order to improve this ratio and optimize the culture method for UCB-MSCs, factorial design were applied to investigate the main influencing factors, including cell inoculate density (ID), combination and dose of cytokines, presence of serum and stromal cells or not. The experimental results indicated that ID was the most significant influencing factor for UCB-MSCs culture when P < 0.1. Higher ID leaded to higher probability of success and better growth for UCB-MSCs. Then were cytokines, which could stimulate the growth of UCB-MSCs effectively. Based on the high cell ID, the optimized culture condition was determined with cytokines IL-3 (15 μg/L) and GM-CSF (5 μg/L) added in the traditional medium of MSCs. Then the probability of obtaining UCB-MSCs can be increased up to 90% from 30% with traditional culture method. Moreover, the characteristics of UCB-MSCs were tested by flow cytometric analysis and multi-lineage differentiation identification for osteoblast, chondrocyte and adipocyte. The results showed that the fibroblast-like cells expressed MSCs surface markers of CD13, CD29, CD105，CD166 and CD44 positively and CD34, CD45 and HLA-DR negatively. Meanwhile the cells could differentiate into osteoblasts, chondrocytes and adipocytes similarly to MSCs derived from bone marrow. In conclusion, an efficient protocol have been developed to culture UCB-MSCs by adding cytokines IL-3 (15 μg/L) and GM-CSF (5 μg/L).

Abstract:A number of recent studies have focused on discovering genetic functional or transcriptional modules by integrating information from the rapidly accumulating large-scale microarray expression datasets. Such studies commonly model each microarray as a co-expression network, and detect the conserved gene co-expression clusters from these co-expression networks. Currently, the commonly used method is mining conserved co-expression clusters directly from a “summary network”, which is obtained by aggregating all the co-expression networks derived from different microarrays. However, this method may generate false conserved clusters, which never occur in any of the original individual co-expression networks. Here a scalable and efficient method were proposed to detect the truly conserved gene co-expression clusters from multiple microarrays. This problem is formulated as mining frequently occurring subgraphs across multiple co-expression networks, and involves three steps: (1) Translating each microarray into co-expression network; (2) Clustering edges which occur in the similar co-expression networks by min-hashing and locality-sensitive hashing techniques to obtain the candidate clusters; (3) Applying graph clustering method to the candidate clusters to detect the conserved co-expressed clusters. This method was applied to yeast microarrays and the results demonstrate that, compared to the previous study, the conserved co-expressed clusters detected by the method were more likely to be functionally homogeneous entities or potential transcriptional modules.

Abstract:Single-chain Fv (scFv) antibody library against SIEA (soluble immature egg antigen) of Schistosoma japonicum was constructed by phage display technology. Specific SIEA26～28 ku scFv was obtained by screening the SIEA scFv library using the natural molecular candidate vaccine, SIEA26～28 ku. The specific scFv fragment was subcloned to prokaryotic expression vector PET32a and induced the expression of soluble specific scFv in high level. Subsequently, specific scFv as a probe was used to screen the cercaria cDNA library of Schistosoma japonicum to get the coding genes of SIEA26～28 ku molecules. Results showed that specific SIEA26～28 ku scFv with high level expression was achieved. The corresponding gene, ribosomal protein S4 was obtained by initially screening the cercaria cDNA library with the specific SIEA26～28 ku scFv. The obtaining of specific SIEA26～28 ku scFv lays the foundation for further screening and identification of the coding genes of SIEA26～28 ku, the natural molecular candidate vaccine against schistosomiasis.

Abstract:Genetic bistable systems are a large class of important biological systems. Bistability, the capacity to achieve two distinct stable steady states in response to a set of external stimuli, arises within biological systems ranging from the λ phage switch in bacteria to cellular signal transduction pathways in mammalian cells. On the other hand, the increasing experimental evidence in the form of bimodal population distribution has indicated that noise plays a very key role in the switching of bistable systems. However, the physiological mechanism underling noise-induced switching behaviors has not been well explored yet. In the previous work, it has been showed that noise can induce coherent switch for a single genetic Toggle switch system. Here the influence of several kinds of noises (including intracellular and extracellular noises) on synchronized switch was investigated for a multicell gene toggle switch network system. It has been found that multiplicative noises resulting from fluctuations of either synthesis or degradation rates and the additive noise within each cell (they altogether are called as intracellular noises) all can induce the synchronized switch, and that there exists an optimal noise intensity such that the synchronized switch is optimally achieved and the amplification factor has the maximal value. On the other hand, the extracellular noises arising from the stochastic fluctuation of the cellular environment, not only brings about the synchronized switch, but also enhances it by suppressing intracellular fluctuations when the intracellular noises are not enough to induce the synchronized switch. Finally, the influence of the diffusive rate of signal molecules affected by noise on the dynamics of the multicellular system was also investigated, showing that the larger the diffusive rate, the better the synchronized switch and the larger the amplification factor.

Abstract:XnBP83 is a clone which had oral toxicity to Helicoverpa. armegera screened from the cosmid library of Xenorhabdus. nematophilus BP. Comsmid XnBP83 was sequenced using a strategy of subcloning and primer-walking DNA sequencing. The inserted sequences of XnBP83 have 38 939 bp including 5 ORFs that are associated to the insecticidal activity: xptA1, xptB1, xptC1, xptA2 and xptD1. The results of sequence analysis showed that: 1) The xptD1 of XnBP83 is incomplete, and shares 99% predicted amino acid sequence identity to that of X. nematophilus PMFI296. 2) The xptA1 of XnBP83 has 7 569 bp nucleotides, encoding 2 520 amino acids which shares 98% identical amino acid residues with that of PMFI296. 3) The xptB1 has 3 051 bp nucleotides, encoding 1 016 amino acids. The XptB1 sequence shares 98% identical amino acid residues with XptB1 of PMFI296. There are 28 different amino acids from 620 to 650 amino acids. 4) The xptC1 of X. nematophilus BP has 4 225 bp, encoding 1 408 amino acids. The XptC1 sequence shares 96% identical amino acid residues with that of X. nematophilus PMFI296. There are 18 different amino acids from 627 to 646 amino acids. The amino acid sequence with TAQRYLAK was inserted to downstream to 232nd amino acid. And 5) The xptA2 has a length of 7 574 bp, encoding 2 524 amino acids. The XptA2 sequence shares 90% identical amino acid residues with that of PMFI296. Two distinctly divergence amino acid regions present from 788 to 855 and from 1 630 to 1 784 amino acids. Oral bioassay of the supernatant and cell sediment of XnBP83 against H. armegera, S. exigua, S. litura, and T. ni showed that XnBP83 had broad-spectrum insecticidal activity.

Abstract:To construct and screen fully human anti-hepatoma single-chain Fv fusion phage libraries, peripheral blood mononuclear cells (PBMCs) of patients with liver cancer were sensitized in vitro and transformed by Epstein-Barr virus (EBV). VH and VL genes were reamplified by PCR and combined to single-chain fragment of variable region (ScFv) genes. ScFv genes were cloned into vector fuse5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library. The library was subjected to three rounds of positive and negative cell panning and enrichment，then was secreened by phage-ELISA. The binding specificity of phage antibodies with hepatoma carcinoma cells was confirmed by immunohistochemistry with cultured cells and tissue sections. Detection of ELISA showed that 4 liver cancer patients'B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell. 6 types of VH genes and 9 types of VL genes were obtained by PCR reamplification then connected with (Gly4Ser)₃ linker to form 54 types of ScFv genes. ScFv genes digested with SfiⅠwere cloned into vector fuse5 and transformed into MC1061 via electroporation. Phage antibody library with sink size being 1.0×10⁸ was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 80%. The library was subjected to three rounds of positive and negative cell panning and enrichment，then was selected by phage-ELISA. After primacy test by HepG2 cell ELISA,179 clones of phage antibody with positive ELISA reaction were picked out of 533 clones.The percentage of positive clones was 33.6%.Though further screened by a panel of cultured cells ELISA,the clone A82 was found to react strongly with HepG2, HEK293, but not with other human tumor cell lines.It also reacted weakly with human hepatic cell line QSG-7701. The results of immunohistochemistry with cultured cells were same as the results of ELISA. A82 was further analyzed after the DNA sequencing.The sequence of A82 was identical. The length of A82 was 742 bp. The VDJ regions of A82 belonged to VH3-23-D2-21-JH6-linker-V4-2-JL2. It can be concluded that fully human anti-hepatoma single-chain Fv fusion phage libraries with sink size being 1.0×10⁸ was constructed by means of phage antibody library technique in combination with in vitro immunization method and EBV transformation technique. By cell ELISA and immunohistochemistry with cultured cells and tissue sections,the clone A82 was confirmed to specific bind with hepatoma carcinoma cells.The ScFv fragment against hepatoma may be further developed and applied to clinical diagnosis and therapy.

Abstract:Seven genes (fabD, fabG, fabH, fabA, fabZ, fabB and fabI) of E.coli fatty acid biosynthetic enzymes were cloned by PCR amplifying and appropriate expression vectors were constructed. Under induction assay expression of plasmid encoded proteins was carried out in strain BL21(DE3) and seven enzymes were purified using Ni-NTA agarose resin. In the absence of [2-¹⁴C] malonyl-CoA fatty acid synthetic reaction was reconstituted in vitro by adding seven enzymes and co-factors. And several model reactions were established for identification of special fatty acid biosynthetic enzymes. Meanwhile Clostridium acetobutylicium FabZ function was characterized by this method.

Abstract:At present, there are many methods for genotype of hepatitis C virus , but not a gold standard. In order to establish the rationale for genotypic determination of optimal region sequence, fifteen complete genome sequences of hepatitis C which had been given the annotation about every region and derived from different country were downloaded from GenBank. Phylogenetic trees on 5′UTR, core, E1, E2 and NS5B region were established. The results demonstrated that genotyping group was not all correct on 5′ UTR region while genotyping groups were wholly correct on core, E1, E2 and NS5B region. Comparing phylogenetic distances on core, E1, E2 and NS5B region with that on complete genome sequence demonstrated that the NS5B area was the best genotyping region instead of the complete genome sequence. In addition, analysis of the molecular evolution on each core region could supply some clues for creating novel genotyping method based on PCR-RFLP.